High throughput screening of genetically modified photosynthetic organisms

ABSTRACT

The present invention provides a method and compositions for high throughput screening of genetically modified photosynthetic organisms for plasmic state. The present invention provides methods of producing one or more proteins, including biomass degrading enzymes in a plant. Also provided are the methods of producing biomass degradation pathways in alga cells, particularly in the chloroplast. Single enzymes or multiple enzymes may be produced by the methods disclosed. The methods disclosed herein allow for the production of biofuel, including ethanol.

CROSS-REFERENCE

This application claims priority to and the benefit of the followingU.S. Provisional Applications: U.S. Ser. No. 60/941,452, filed Jun. 1,2007, entitled of “USE OF PHOTOSYNTHETIC ORGANISMS TO GENERATE BIOFUELS”by Mayfield et al; U.S. Ser. No. 61/070,384, filed Mar. 20, 2008,entitled “USE OF GENETICALLY MODIFIED ORGANISMS TO GENERATE BIOMASSDEGRADING ENZYMES” by Mayfield et al.; and U.S. Ser. No. 61/070,437filed Mar. 20, 2008, entitled “HIGH THROUGHPUT SCREENING OF GENETICALLYMODIFIED PHOTOSYNTHETIC ORGANISMS” by Mayfield et al. Each of theseprior applications is incorporated herein by reference.

INCORPORATION BY REFERENCE

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

BACKGROUND OF THE INVENTION

Fuel is becoming increasingly more expensive. Also, fuel refinery isassociated with the generation of pollutants and global warming. Thereis an increasing need in the industry to find cheaper, safer, and moreenvironmentally unharmful ways to generate fuels. The development ofmeans to produce fuel from biological material is an essential componentof the future energy landscape. One of the most important elements inthe production of fuel from biologic materials is the ability to digestor reduce certain molecular structures, such as cellulose, to molecularspecies recognizable as substrate for fuel generating processes, such asfermentation.

Molecular biology and genetic engineering hold promise for theproduction of large quantities of biologically active molecules that canbe used to produce such fuels. For example, production of enzymescapable of breaking down organic materials into fuels hold promise toaddress the increasing needs for alternative fuels. A primary advantageof using genetic engineering techniques for producing such enzymes isthat the methods allow for the generation of large amounts of a desiredprotein. In many cases, the only other way to obtain sufficientquantities of biological materials from non-engineered secretion sourcesis by purifying the naturally occurring biological material from cellsof an organism that produce the agent. Thus, prior to the advent ofgenetic engineering, enzymes capable of degrading organic materialscould only be isolated by growing the organism, typically a bacterial orfungal species, in large quantities and extracting the protein. Suchprocedures are often complex and economically prohibitive for use infuel production.

Although genetic engineering provides a means to produce large amountsof a biological material, particularly proteins and nucleic acids, thereare limitations to currently available methods. Bacteria provide anenvironment suitable to the production of such enzymes; however,byproducts produced by some bacteria would contaminate fuel sources.Thus, even where bacteria can be used to produce the biologicalmaterial, additional steps such as purification or refining may berequired to obtain biologically active material and/or bio-fuel.Furthermore, the use of non-photosynthetic systems requires the additionof costly sugar or other organic carbon sources to feed the recombinantorganism. Additionally, there is typically a large capital investmentassociated with building fermenters.

Recombinant proteins also can be produced in eukaryotic cells,including, for example, fungi, insect cells and mammalian cells, whichmay provide the necessary environment to process an expressed proteininto a biologically active agent. However, these systems typicallysuffer from the same cost prohibitions (sugar/organic carbon sources andfermenters). Thus, a need exists for methods to conveniently produceenzymes that are biologically active, can produce large quantities ofenzymes and/or provide a host organism which is compatible withproduction of degradative enzymes.

SUMMARY OF THE INVENTION

Presented herein are compositions and methods for the production ofbiomass degrading enzymes and biofuels. The inventions disclosed hereinprovide novel methods for the production of biomass degrading enzymes,typically in genetically modified photosynthetic organisms such as algaeand cyanobacteria. Also presented herein are compositions and methodsfor transforming photosynthetic organisms and methods of screeningtransformants.

Accordingly, one aspect of the present invention provides a vectorcomprising a nucleic acid encoding a biomass degrading enzyme and apromoter configured for expression of the nucleic acids in anon-vascular photosynthetic organism. Vectors of the present inventionmay contain nucleic acids encoding more than one biomass degradingenzyme and, in other instances, may contain nucleic acids encodingpolypeptides which covalently link biomass degrading enzymes. Biomassdegrading enzymes may include cellulolytic enzymes, hemicellulolyticenzymes and ligninolytic enzymes. More specifically, the biomassdegrading enzymes may be exo-β-glucanase, endo-β-glucanase,β-glucosidase, endoxylanase, or lignase. Nucleic acids encoding thebiomass degrading enzymes may be derived from fungal or bacterialsources, for example, those encoding exo-β-glucanase in Trichodermaviride, exo-β-glucanase in Trichoderma reesei, exo-β-glucanase inAspergillus aculeatus, endo-β-glucanase in Trichoderma reesei,endo-β-glucanase in Aspergillus niger, β-glucosidase in Trichodermareesei, β-glucosidase in Aspergillus niger endoxylanase in Trichodermareesei, and endoxylanase in Aspergillus niger. Other nucleic acidsencoding biomass degrading enzymes may be homologous to the genes fromthese organisms

A vector of the present invention may also contain a selectable marker,allowing for direct screening of transformed organisms. The vectors ofthe present invention may be capable of stable transformation ofmultiple photosynthetic organisms, including, but not limited to,photosynthetic bacteria (including cyanobacteria), cyanophyta,prochlorophyta, rhodophyta, chlorophyta, heterokontophyta, tribophyta,glaucophyta, chlorarachniophytes, euglenophyta, euglenoids, haptophyta,chrysophyta, cryptophyta, cryptomonads, dinophyta, dinoflagellata,pyrmnesiophyta, bacillariophyta, xanthophyta, eustigmatophyta,raphidophyta, phaeophyta, and phytoplankton. Other vectors of thepresent invention are capable of stable transformation of C.reinhardtii, D. salina or H. pluvalis. Still other vectors containnucleic acids which are biased to an organism's (e.g., C. reinhardtii)codon preference. Specific vectors of the present invention containsequences provided herein (SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21,SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO.26, or SEQ ID NO. 27).

Host cells comprising the vectors of the present invention are alsoprovided. In some instances, the host cell is a non-vascularphotosynthetic organism, for example, an organism classified asphotosynthetic bacteria (including cyanobacteria), cyanophyta,prochlorophyta, rhodophyta, chlorophyta, heterokontophyta, tribophyta,glaucophyta, chlorarachniophytes, euglenophyta, euglenoids, haptophyta,chrysophyta, cryptophyta, cryptomonads, dinophyta, dinoflagellata,pyrmnesiophyta, bacillariophyta, xanthophyta, eustigmatophyta,raphidophyta, phaeophyta, and phytoplankton. A host cell of the presentinvention may also be a microalga species including, but not limited to,C. reinhardtii, D. salina or H. pluvalis. In other instances, the hostcell may be one or more cells of a multicellular photosyntheticorganism. For some embodiments, the host cell may be grown in theabsence of light and/or in the presence of an organic carbon source.

The present invention also provides compositions containing one or moreexogenous biomass degrading enzymes derived from one or morenon-vascular photosynthetic organisms. In some instances, thesecompositions may also contain elements of the non-vascularphotosynthetic organisms. The ratio (w/w) of enzymes to elements of theorganisms may be at least 1:10, or the elements may be found only intrace amounts. Some of the compositions comprise at least one of thefollowing enzymes: exo-β-glucanase, endo-β-glucanase, β-glucosidase,endoxylanase, and/or lignase; where the enzyme(s) is isolated from oneor more of the following organisms: C. reinhardtii, D. salina, H.pluvalis, photosynthetic bacteria (including cyanobacteria), cyanophyta,prochlorophyta, rhodophyta, chlorophyta, heterokontophyta, tribophyta,glaucophyta, chlorarachniophytes, euglenophyta, euglenoids, haptophyta,chrysophyta, cryptophyta, cryptomonads, dinophyta, dinoflagellata,pyrmnesiophyta, bacillariophyta, xanthophyta, eustigmatophyta,raphidophyta, phaeophyta, and phytoplankton. For some embodiments, theorganism may be grown in the absence of light and/or in the presence ofan organic carbon source.

The present invention also provides a composition containing a pluralityof vectors each of which encodes a different biomass degrading enzymeand a promoter for expression of said biomass degrading enzymes in achloroplast. Such compositions may contain multiple copies of aparticular vector encoding a particular enzyme. In some instances, thevectors will contain nucleic acids encoding cellulolytic,hemicellulolytic and/or ligninolytic enzymes. More specifically, theplurality of vectors may contain vectors capable of expressingexo-β-glucanase, endo-β-glucanase, β-glucosidase, endoxylanase and/orlignase. Some of the vectors of this embodiment are capable of insertioninto a chloroplast genome and such insertion can lead to disruption ofthe photosynthetic capability of the transformed chloroplast. Insertionof other vectors into a chloroplast genome does not disruptphotosynthetic capability of the transformed chloroplast. Some vectorsprovide for expression of biomass degrading enzymes which aresequestered in a transformed chloroplast. Still other vectors maycontain specific sequences provided herein (SEQ ID NO. 19, SEQ ID NO.20, SEQ ID NO. 21, SEQ ID NO. 22, or SEQ ID NO. 23, SEQ ID NO. 24, SEQID NO. 25, SEQ ID NO. 26, or SEQ ID NO. 27). The present invention alsoprovides an algal cell containing the vector compositions describedabove and specifically provides C. reinhardtii, D. salina or H. pluvaliscells containing the vector compositions. For some embodiments, the cellmay be grown in the absence of light and/or in the presence of anorganic carbon source.

Another vector of the present invention encodes a plurality of distinctbiomass degrading enzymes and a promoter for expression of the biomassdegrading enzymes in a non-vascular photosynthetic organism. The biomassdegrading enzymes may be one or more of cellulollytic, hemicellulolyticor ligninolytic enzymes. In some vectors, the plurality of distinctbiomass degrading enzymes is two or more of exo-β-glucanase,endo-β-glucanase, β-glucosidase, lignase and endoxylanase. In someembodiments, the plurality of enzymes is operatively linked. In otherembodiments, the plurality of enzymes is expressed as a functionalprotein complex. Insertion of some vectors into a host cell genome doesnot disrupt photosynthetic capability of the organism. Vectors encodinga plurality of distinct enzymes, may lead to production of enzymes whichare sequestered in a chloroplast of a transformed organism. The presentinvention also provides an algal cell or cyanobacterial cell transformedwith a vector encoding a plurality of distinct enzymes. In someinstances, the algal cell is C. reinhardtii, D. salina or H. pluvalis.In other instances, the cyanobacterial cell is a species of the genusSynechocystis or the genus Synechococcus or the genus Athrospira. Forsome embodiments, the organism may be grown in the absence of lightand/or in the presence of an organic carbon source.

Yet another aspect of the present invention provides a geneticallymodified chloroplast producing one or more biomass degrading enzymes.Such enzymes may be cellulolytic, hemicellulolytic or ligninolyticenzymes, and more specifically, may be an exo-β-glucanase, anendo-β-glucanase, a β-glucosidase, an endoxylanase, a lignase and/orcombinations thereof. The one or more enzymes are be sequestered in thechloroplast in some embodiments. The present invention also providesphotosynthetic organisms containing the genetically modifiedchloroplasts of the present invention.

Yet another aspect provides a method for preparing a biomass-degradingenzyme. This method comprises the steps of (1) transforming aphotosynthetic, non-vascular organism to produce or increase productionof said biomass-degrading enzyme and (2) collecting thebiomass-degrading enzyme from said transformed organism. Transformationmay be conducted with a composition containing a plurality of differentvectors encoding different biomass degrading enzymes. Transformation mayalso be conducted with a vector encoding a plurality of distinct biomassdegrading enzymes. Any or all of the enzymes may be operatively linkedto each other. In some instances, a chloroplast is transformed. Thismethod of the invention may have one or more additional steps,including: (a) harvesting transformed organisms; (b) drying transformedorganisms; (c) harvesting enzymes from a cell medium; (d) mechanicallydisrupting transformed organisms; or (e) chemically disruptingtransformed organisms. The method may also comprise further purificationof an enzyme through performance liquid chromatography. In someinstances the transformed organism is an alga or a photosyntheticbacteria, e.g., cyanobacteria. For some embodiments, the organism may begrown in the absence of light and/or in the presence of an organiccarbon source.

Still another method of the present invention allows for preparing abiofuel. One step of this method includes treating a biomass with one ormore biomass degrading enzymes derived from a photosynthetic,non-vascular organism for a sufficient amount of time to degrade atleast a portion of said biomass. The biofuel produced may be ethanol.The enzymes of this method may contain at least traces of saidphotosynthetic non-vascular organism from which they are derived.Additionally, the enzymes useful for some embodiments of this methodinclude cellulolytic, hemicellulolytic and ligninolytic enzymes.Specific enzymes useful for some aspects of this method includeexo-β-glucanase, endo-β-glucanase, β-glucosidase, endoxylanase, and/orlignase. The organisms of this method may include photosyntheticbacteria (including cyanobacteria), cyanophyta, prochlorophyta,rhodophyta, chlorophyta, heterokontophyta, tribophyta, glaucophyta,chlorarachniophytes, euglenophyta, euglenoids, haptophyta, chrysophyta,cryptophyta, cryptomonads, dinophyta, dinoflagellata, pyrmnesiophyta,bacillariophyta, xanthophyta, eustigmatophyta, raphidophyta, phaeophyta,and phytoplankton. Other organisms used for this method are microalgaeincluding, but not limited to C. reinhardtii, D. salina and H. pluvalis.For some embodiments, the organism may be grown in the absence of lightand/or in the presence of an organic carbon source. Multiple types ofbiomass including agricultural waste, paper mill waste, corn stover,wheat stover, soy stover, switchgrass, duckweed, poplar trees,woodchips, sawdust, wet distiller grain, dray distiller grain, humanwaste, newspaper, recycled paper products, or human garbage may betreated with this method of the invention. Biomass may also be derivedfrom a high-cellulose content organism, such as switchgrass or duckweed.The enzyme(s) used in this method may be liberated from the organism andthis liberation may involve chemical or mechanical disruption of thecells of the organism. In an alternate embodiment, the enzyme(s) aresecreted from the organism and then collected from a culture medium. Thetreatment of the biomass may involve a fermentation process, which mayutilize a microorganism other than the organism which produced theenzyme(s). In some instances the non-vascular photosynthetic organismmay be added to a saccharification tank. This method of the inventionmay also comprise the step of collecting the biofuel. Collection may beperformed by distillation. In some instances, the biofuel is mixed withanother fuel.

An additional method of the present invention provides for making atleast one biomass degrading enzyme by transforming a chloroplast to makea biomass degrading enzyme. The biomass degrading enzyme may be acellulolytic enzyme, a hemicellulolytic enzyme, or a ligninolyticenzyme, and specifically may be exo-β-glucanase, endo-β-glucanase,β-glucosidase, endoxylanase, or lignase. In some instances, the biomassdegrading enzyme is sequestered in the transformed chloroplast. Themethod may further involve disrupting, via chemical or mechanical means,the transformed chloroplast to release the biomass degrading enzyme(s).In some instances, multiple enzymes will be produced by a transformedchloroplast. The biomass degrading enzymes may be of fungal or bacterialorigin, for example, exo-β-glucanase, endo-β-glucanase, β-glucosidase,endoxylanase, lignase, or a combination thereof.

Yet another method of the present invention provides for screening atransformed non-vascular photosynthetic organism, by amplifying a firstnucleic acid sequence from a chloroplast of said organism and amplifyinga second nucleic acid sequence from said chloroplast of said organismand determining the plasmic state of said organism based on results fromamplification of said first sequence and second sequence. In someinstances the first and second amplifying step is performedsimultaneously. The first nucleic acid sequence may be an endogenouschloroplast genome sequence and the second nucleic acid sequence may beat least partially from an exogenous nucleic acid. In some instances, athird nucleic acid sequence from the chloroplast may be amplified as acontrol. This third nucleic acid sequence may be a wild-type sequencethat remains intact after integration of exogenous nucleic acid(s).Where this third nucleic acid is amplified, such amplification may beperformed concurrently with the first or second amplifying step, or allthree amplifications may be performed concurrently. For amplificationsof this method, the specific primers provided herein—SEQ ID NO. 1, SEQID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ IDNO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 1, SEQ IDNO. 12, SEQ ID NO. 13, SEQ ID NO. 14, or SEQ ID NO. 15—may be utilized.Amplification of the first and/or second nucleic acid may utilize morethan thirty cycles of PCR. In some instances, determining the plasmicstate is performed by visual analysis of products from the amplifyingsteps. One or more amplifications may be performed using real-time orquantitative PCR.

The plasmic state determined by this method may be homoplasmy and theorganism tested may be a microalga, specifically, one of the microalgaspecies C. reinhardtii, D. salina or H. pluvalis. In this method, theorganism may contain an exogenous nucleic acid which contains a gene ofinterest and a selectable marker. The gene of interest may encode abiomass degrading enzyme, for example a cellulolytic, hemicellulolyticor lignolytic enzyme. Specifically, the biomass degrading enzyme may beexo-β-glucanase, endo-β-glucanase, β-glucosidase, endoxylanase orlignase. Additionally, the exogenous nucleic acid may be one of thenucleic acids specifically provided herein—SEQ ID NO. 19, SEQ ID NO. 20,SEQ ID NO. 21, SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO.25, SEQ ID NO. 26, SEQ ID NO. 27, SEQ ID NO. 28, SEQ ID NO. 29, SEQ IDNO. 30, or SEQ ID NO. 31.

The present invention also provides a non-vascular photosyntheticorganism containing a homoplasmic chloroplast population, where thechloroplast population comprises an exogenous nucleic acid and where thehomoplasmic state of the chloroplast population is determined by atleast two different PCR reactions. In some instances, the chloroplastpopulation is more than one chloroplast. The non-vascular photosyntheticorganism may be a microalga, specifically one of the species C.reinhardtii, D. salina or H. pluvalis. The organism may be screenedusing at least two different PCR reactions performed simultaneously.These PCR reactions may utilize one of the specific primers disclosedherein—SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ IDNO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ IDNO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 14, orSEQ ID NO. 15. The PCR reactions may utilize more than thirty cycles.

The organism may contain an exogenous nucleic acid comprising at leastone gene of interest and a selectable marker. This gene may encode abiomass degrading enzyme, specifically a cellulolytic, hemicellulolyticor ligninolytic enzyme. Even more specifically, the biomass degradingenzyme may be exo-β-glucanase, endo-β-glucanase, β-glucosidase,endoxylanase or lignase. The exogenous nucleic acid present in thisorganism of the present invention may be on of the nucleic acidsspecifically described herein—SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO.21, SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 25, SEQ IDNO. 26, SEQ ID NO. 27, SEQ ID NO. 28, SEQ ID NO. 29, SEQ ID NO. 30, orSEQ ID NO. 31.

Another method is provided herein for producing a genetically-modifiedhomoplasmic non-vascular photosynthetic organism. This method involvestransforming at least one chloroplast of the organism with an exogenousnucleic acid, amplifying a first nucleic acid sequence and a secondnucleic acid sequence, and determining the plasmic state of the organismbased on results from the amplifying step. The first and second nucleicacid sequences may be within the chloroplast genome. Additionally, thefirst nucleic acid sequence may be an endogenous chloroplast sequence.The second nucleic acid sequence may be at least partially from theexogenous nucleic acid. This method may also involve amplifying a thirdnucleic acid sequence from the chloroplast as a control. In someinstances the third nucleic acid is a wild-type sequence that remainsintact after integration of an exogenous nucleic acid. This method mayinvolve PCR using one of the specifically disclosed primers herein—SEQID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ IDNO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ IDNO. 11, SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 14, or SEQ ID NO. 15.Amplification of the first and second nucleic acid sequences may utilizemore than thirty cycles of PCR. The determination of plasmic state usingthis method may involve visual analysis of the products of theamplifying step(s).

The plasmic state determined by this method may be homoplasmy and theorganism may be a microalga, specifically one of the species C.reinhardtii, D. salina or H. pluvalis. The exogenous nucleic acid maycontain at least one gene of interest and a selectable marker. In someinstances, the gene of interest encodes a biomass degrading enzyme,specifically a cellulolytic, hemicellulolytic or ligninolytic enzyme.Even more specifically the biomass degrading enzyme may beexo-β-glucanase, endo-β-glucanase, β-glucosidase, endoxylanase orlignase. Moreover, the exogenous nucleic acid may be one specificallydescribed herein—SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO.22, SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO. 26, SEQ IDNO. 27, SEQ ID NO. 28, SEQ ID NO. 29, SEQ ID NO. 30, or SEQ ID NO. 31.

Another embodiment of the present invention is a kit for determiningplasmic state of a genetically-modified non-vascular photosyntheticorganism. Such a kit may contain amplification primer(s) for amplifyinga first nucleic acid sequence of a chloroplast genome corresponding toan endogenous sequence and amplification primer(s) for amplifying asecond nucleic acid sequence of a chloroplast genome that is anintroduced or non-naturally occurring sequence. A kit may also contain aPCR buffer and/or amplification primer(s) for amplifying a controlnucleic acid sequence. A kit may contain one or more of the PCR primersspecifically disclosed herein—SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3,SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8,SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO.13, SEQ ID NO. 14, or SEQ ID NO. 15. The primer(s) for amplifying afirst nucleic acid sequence in a kit of the present invention, may bindto at least a portion of a psbA 5′UTR, a psbA coding sequence, an psbC5′ UTR, a psbD 5′ UTR, an atpA 5′ UTR, or a 3HB locus. In someinstances, at least one of the primer(s) for amplifying a second nucleicacid sequence will bind to at least a portion of a sequence encoding abiomass degrading enzyme, such as a cellulolytic, hemicellulolytic orligninolytic enzyme. Specific biomass degrading enzymes encoded by thesecond nucleic acid may be exo-β-glucanase, endo-β-glucanase,β-glucosidase, endoxylanase or lignase. The primers may amplify at leasta portion of one or more of the sequences specifically disclosedherein—SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22, SEQID NO. 23, SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27,SEQ ID NO. 28, SEQ ID NO. 29, SEQ ID NO. 30, or SEQ ID NO. 31.Additionally, the kit may contain instructions for use.

SUMMARY OF THE FIGURES

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 illustrates transformation of alga cells, selection,confirmation, and scaling of production of enzymes.

FIG. 2 illustrates two constructs for insertion of a gene into achloroplast genome.

FIG. 3 illustrates primer pairs for PCR screening of transformants andexpected band profiles for wild-type, heteroplasmic and homoplasmicstrains.

FIG. 4 illustrates results from PCR screening and Western blot analysisof endo-β-glucanase transformed C. reinhardtii clones.

FIG. 5 illustrates results from PCR screening and Western blot analysisof exo-β-glucanase transformed C. reinhardtii clones.

FIG. 6 illustrates results from PCR screening and Western blot analysisof β-glucosidase transformed C. reinhardtii clones.

FIG. 7 illustrates results from PCR screening and Western blot analysisof endoxylanase transformed C. reinhardtii clones.

FIG. 8 illustrates determination of the level of endo-β-glucanaseprotein produced by transformed C. reinhardtii clones.

FIG. 9 is a graphic representation of an embodiment of the presentinvention, showing generalized constructs for insertion of multiplegenes into a chloroplast genome.

FIG. 10 illustrates results from PCR screening and Western blot analysisof endo-β-glucanase transformed C. reinhardtii clones.

FIG. 11 illustrates results from PCR screening and Western blot analysisof β-glucosidase transformed C. reinhardtii clones.

FIG. 12 is a graphic representation of two exogenous DNA constructs forinsertion into a chloroplast genome.

FIG. 13 is a graphic representation of two exogenous DNA constructs forinsertion into a cyanobacterial genome.

FIG. 14 illustrates results from PCR screening and Western blot analysisof endo-β-glucanase transformed C. reinhardtii clones.

FIG. 15 illustrates results from PCR screening and Western blot analysisof endoxylanase transformed C. reinhardtii clones.

FIG. 16 illustrates results from PCR screening and Western blot analysisof exo-β-glucanase transformed C. reinhardtii clones.

FIG. 17 illustrates activity of bacterially-produced biomass degradingenzymes.

DETAILED DESCRIPTION OF THE INVENTION

Technical and scientific terms used herein have the meanings commonlyunderstood by one of ordinary skill in the art to which the instantinvention pertains, unless otherwise defined. Reference is made hereinto various materials and methodologies known to those of skill in theart. Standard reference works setting forth the general principles ofrecombinant DNA technology include Sambrook et al., “Molecular Cloning:A Laboratory Manual”, 2d ed., Cold Spring Harbor Laboratory Press,Plainview, N.Y., 1989; Kaufman et al., eds., “Handbook of Molecular andCellular Methods in Biology and Medicine”, CRC Press, Boca Raton, 1995;and McPherson, ed., “Directed Mutagenesis: A Practical Approach”, IRLPress, Oxford, 1991. Standard reference literature teaching generalmethodologies and principles of yeast genetics useful for selectedaspects of the invention include: Sherman et al. “Laboratory CourseManual Methods in Yeast Genetics”, Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y., 1986 and Guthrie et al., “Guide to Yeast Geneticsand Molecular Biology”, Academic, New York, 1991.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimits of that range is also specifically disclosed. Each smaller rangebetween any stated value or intervening value in a stated range and anyother stated or intervening value in that stated range is encompassed.The upper and lower limits of these smaller ranges can independently beincluded or excluded in the range, and each range where either, neitheror both limits are included in the smaller ranges is also encompassed,subject to any specifically excluded limit in the stated range. Wherethe stated range includes one or both of the limits, ranges excludingeither or both of those included limits are also included.

The present invention relates to the production of enzymes, e.g.,biomass degrading enzymes, by genetically modified organisms. Anotheraspect of the present invention relates to compositions and methods forusing biologic material to create products, such as ethanol, usingbiomass degrading enzymes produced by photosynthetic microorganisms,such as, but not limited to, algae. Typically, photosynthetic organismsdo not possess all of the necessary enzymes to degrade biomass. Thepresent invention takes advantage of the ability to introduce exogenousnucleic acids into algal cells, and particularly into the chloroplastsof those cells. One advantage of using molecular biology and geneticengineering to create enzyme-expressing and/or enzymaticpathway-expressing algal strains is the potential for the production oflarge quantities of active enzymes.

One approach to construction of a genetically manipulated strain of algais diagramed as a flow chart in FIG. 1. As can be seen, alga cells(e.g., Chlamydomonas reinhardti, Dunaliella salina, Hematococcuspluvalis) are transformed with a nucleic acid which encodes a gene ofinterest, typically a biomass degrading enzyme. In some embodiments, atransformation may introduce nucleic acids into any plastid of the hostalga cell (e.g., chloroplast). Transformed cells are typically plated onselective media following introduction of exogenous nucleic acids. Thismethod may also comprise several steps for screening. Initially, ascreen of primary transformants is typically conducted to determinewhich clones have proper insertion of the exogenous nucleic acids.Clones which show the proper integration may be patched and re-screenedto ensure genetic stability. Such methodology ensures that thetransformants contain the genes of interest. In many instances, suchscreening is performed by polymerase chain reaction (PCR); however, anyother appropriate technique known in the art may be utilized. Manydifferent methods of PCR are known in the art (e.g., nested PCR, realtime PCR). For any given screen, one of skill in the art will recognizethat PCR components may be varied to achieve optimal screening results.For example, magnesium concentration may need to be adjusted upwardswhen PCR is performed on disrupted alga cells as many such organismshave magnesium chelators. In such instances, magnesium concentration mayneed to be adjusted upward, or downward (compared to the standardconcentration in commercially available PCR kits) by 0.1, 0.2, 0.3, 0.4,0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8,1.9, or 2.0 mM. Thus, after adjusting, final magnesium concentration ina PCR reaction may be, for example 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3,1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7,2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5 mM or higher. Particular examplesare utilized in the examples described herein; however, one of skill inthe art will recognize that other PCR techniques may be substituted forthe particular protocols described. Following screening for clones withproper integration of exogenous nucleic acids, typically clones arescreened for the presence of the encoded protein. Protein expressionscreening typically is performed by Western blot analysis and/or enzymeactivity assays.

Following confirmation of nucleic acid integration and/or proteinexpression, selected clones may be scaled up for production of biofuelsthrough biomass degradation, first in smaller volumes of 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 6061, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, 99, 100 or more liters. Following initial scaling up, largerscale degradation of biomass may be performed in larger quantities. Oneexample of a partially closed bioreactor system is shown in FIG. 1, step6. However, growth of the transformed strains for biomass degradationand/or biofuel production can also be accomplished in man-madestructures such as ponds, aqueducts, reservoirs and/or landfills.Alternately, the strains can also be grown directly in naturallyoccurring bodies of water, e.g., in ocean, sea, lakes, or rivers. Insome cases, transformed strains are grown near ethanol production plantsor other facilities. Alternately, the biomass degrading cells may begrown near regions (e.g., electrical generating plants, concrete plants,oil refineries, other industrial facilities, cities, highways, etc.)generating CO₂. As such, the methods disclosed herein furthercontemplate business methods for selling carbon credits to ethanolplants or other facilities or regions generating CO₂ while making orcatalyzing the production of fuels by growing one or more of themodified organisms described herein near the ethanol production plant.

The present invention contemplates making biomass degrading enzymes bytransforming host cells (e.g., alga cells such as C. reinhardtii, D.salina, H. pluvalis and cyanobacterial cells) and/or organismscomprising host cells with nucleic acids encoding one or more differentbiomass degrading enzymes (e.g., cellulolytic enzymes, hemicellulolyticenzymes, xylanases, lignases and cellulases). In some embodiments, asingle enzyme may be produced. For example, a cellulase which breaksdown pretreated cellulose fragments into double glucose molecules(cellobiose) or a cellulase which splits cellobiose into glucose, may beproduced.

Some host cells may be transformed with multiple genes encoding one ormore enzymes. For example, a single transformed cell may containexogenous nucleic acids encoding an entire biodegradation pathway. Oneexample of a pathway might include genes encoding an exo-β-glucanase(acts on the cellulose end chain), an endo-β-glucanase (acts on theinterior portion of a cellulose chain), β-glucosidase (avoids reactioninhibitors by/degrades cellobiose), and endoxylanase (acts onhemicellulose cross linking). Such cells transformed with entirepathways and/or enzymes extracted from them, can degrade certaincomponents of biomass. Constructs may contain multiple copies of thesame gene, and/or multiple genes encoding the same enzyme from differentorganisms, and/or multiple genes with mutations in one or more parts ofthe coding sequences.

Alternately, biomass degradation pathways can be created by transforminghost cells with the individual enzymes of the pathway and then combiningthe cells producing the individual enzymes. This approach allows for thecombination of enzymes to more particularly match the biomass ofinterest by altering the relative ratios of the multiple transformedstrains. For example, two times as many cells expressing the firstenzyme of a pathway may be added to a mix where the first step of thereaction pathway is the limiting step.

Following transformation with enzyme-encoding constructs, the host cellsand/or organisms are grown. The biomass degrading enzymes may becollected from the organisms/cells. Collection may be by any means knownin the art, including, but not limited to concentrating cells,mechanical or chemical disruption of cells, and purification of enzymesfrom cell cultures and/or cell lysates. Cells and/or organisms can begrown and then the enzyme(s) collected by any means. One method ofextracting the enzyme is by harvesting the host cell or a group of hostcells and then drying the host cell(s). The enzyme(s) from the driedhost cell(s) are then harvested by crushing the cells to expose theenzyme. The whole product of crushed cells is then used to degradebiomass. Many methods of extracting proteins from intact cells are wellknown in the art, and are also contemplated herein (e.g., introducing anexogenous nucleic acid construct in which an enzyme-encoding sequence isoperably linked to a sequence encoding a secretion signal—excretedenzyme is isolated from the growth medium). Following extraction of theprotein from the cells/organisms and/or the surrounding medium, theprotein may be purified from the crude extract such that the enzyme maycomprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 30, 40, 50, 60, 70, 80, 90, 95, 99 percent or higher of thetotal protein. Purification steps include, but are not limited to, usingHPLC, affinity columns, and antibody-based purification methods.

Extracting and utilizing the biomass-degrading enzyme can also beaccomplished by expressing a vector containing nucleic acids that encodea biomass production-modulation molecule in the host cell. In thisembodiment, the host cell produces the biomass, and also produces abiomass-degrading enzyme. The biomass-degrading enzyme can then degradethe biomass produced by the host cell. In some instances, vector usedfor the production of a biomass-degrading enzyme may not be continuouslyactive. Such vectors can comprise one or more activatable promoters andone or more biomass-degrading enzymes. Such promoters activate theproduction of biomass-degrading enzymes, for example, after the biomasshas grown to sufficient density or reached certain maturity.

A method of the invention can be performed by introducing a recombinantnucleic acid molecule into a chloroplast, wherein the recombinantnucleic acid molecule includes a first polynucleotide, which encodes atleast one polypeptide (i.e., 1, 2, 3, 4, or more). In some embodiments,a polypeptide is operatively linked to a second, third, fourth, fifth,sixth, seventh, eighth, ninth, tenth and/or subsequent polypeptide. Forexample, several enzymes in a biodegradation pathway may be linked,either directly or indirectly, such that products produced by one enzymein the pathway, once produced, are in close proximity to the next enzymein the pathway.

For transformation of chloroplasts, one major benefit of the presentinvention is the utilization of a recombinant nucleic acid constructwhich contains both a selectable marker and one or more genes ofinterest. Typically, transformation of chloroplasts is performed byco-transformation of chloroplasts with two constructs: one containing aselectable marker and a second containing the gene(s) of interest.Screening of such transformants is laborious and time consuming formultiple reasons. First, the time required to grow some transformedorganisms is lengthy. Second, transformants must be screened both forpresence of the selectable marker and for the presence of the gene(s) ofinterest. Typically, secondary screening for the gene(s) of interest isperformed by Southern blot (see, e.g. PCT/US2007/072465).

Constructs of the current invention (FIG. 2, FIG. 9 and FIG. 12), allowfor a PCR-based screening method in which transformants can be screenedusing a combination of primers specific for the insert and wild-typesequences (FIG. 3, lanes: G—gene specific reaction; C—control reaction;WT—wild type reaction; M—multiplex). This methodology provides a rapidscreening process and advances over older techniques. For example,selection of transformants receiving unlinked markers inherently yieldsa lower percentage of clones with the transgenes. Because of this, thelikelihood of obtaining homoplasmic lines from a primary transformationis low. By linking the marker and the gene(s) of interest, thelikelihood of obtaining transgenic clones with the transgene, especiallyhomoplasmic clones, is improved on the first pass. Specific PCRprotocols for screening transformants are detailed in the Examplesbelow, but one of skill in the art will recognize that these protocolsmay be altered to provide quantitative analysis of transformants. Forexample, different ratios of primers for a particular reaction may beutilized to compare insert copy number to a control reaction. Suchvariation may be performed where the multiplex reactions (FIG. 3, row M)are run concurrently or separately.

Determination of insert copy number may be important in some embodimentswhere an optimal level of expression of the exogenous gene(s) ofinterest is, in part, determined by gene copy number. For example,transformation of an alga host cell (e.g., C. reinhardtii, D. salina, H.pluvalis) which results in incorporation of the exogenous nucleic acidin less than half of the copies of the chloroplast genomes in a cell mayyield little or no detectable expression of the gene(s) of interest.Alternately, incorporation of exogenous nucleic acid in all the copiesof the chloroplast genomes in a cell may yield little or no detectableexpression of the gene(s) of interest where there are few initial copiesof the genome (e.g., quantitative PCR analysis will allow for exclusionof homoplasmic clones which have low insert copy number, and thus maynot have sufficiently high production of the gene and/or polypeptide ofinterest). In other embodiments, there may be an optimum level ofincorporation of exogenous nucleic acid. In some instances, exogenousDNA may encode a protein which, whether through transcriptional,translational, or other control mechanisms, is optimally produced whenit is present in a particular range of copy number. Thus, determiningthe copy number of such exogenous DNA, for example by quantitative PCR,may allow selection and/or production of transformed organisms whichproduce protein(s) of interest at an efficient level.

Additionally, recombinant nucleic acid molecules of the presentinvention may be operatively linked to a second and/or subsequentnucleotide sequence. For example, the nucleotide sequences encodingenzymes of a biodegradation pathway may be operatively linked such thatexpression of these sequences may be controlled with a single inducingstimulus or controlled by a single transcriptional activator. Suchsystems are similar to bacterial operons (e.g., the Escherischia coliLac operon). However, these groupings of operatively linked nucleotidesequences in the present invention are synthetic and designed tofunction in plant plastids, preferably are incorporated into thechloroplast genome.

As used herein, the term “operatively linked” means that two or moremolecules are positioned with respect to each other such that they actas a single unit and affect a function attributable to one or bothmolecules or a combination thereof. For example, a polynucleotideencoding a polypeptide can be operatively linked to a transcriptional ortranslational regulatory element, in which case the element confers itsregulatory effect on the polynucleotide similarly to the way in whichthe regulatory element would affect a polynucleotide sequence with whichit normally is associated with in a cell. A first polynucleotide codingsequence also can be operatively linked to a second (or more) codingsequence such that a chimeric polypeptide can be expressed from theoperatively linked coding sequences. The chimeric polypeptide producedfrom such a construct can be a fusion protein, in which the two (ormore) encoded peptides are translated into a single polypeptide, i.e.,are covalently bound through a peptide bond, either directly or with ashort spacer region.

In chloroplasts, regulation of gene expression generally occurs aftertranscription, and often during translation initiation. This regulationis dependent upon the chloroplast translational apparatus, as well asnuclear-encoded regulatory factors (see Barkan and Goldschmidt-Clermont,Biochemie 82:559-572, 2000; Zerges, Biochemie 82:583-601, 2000). Thechloroplast translational apparatus generally resembles that inbacteria; chloroplasts contain 70S ribosomes; have mRNAs that lack 5′caps and generally do not contain 3′ poly-adenylated tails (Harris etal., Microbiol. Rev. 58:700-754, 1994); and translation is inhibited inchloroplasts and in bacteria by selective agents such aschloramphenicol.

Some methods of the present invention take advantage of properpositioning of a ribosome binding sequence (RBS) with respect to acoding sequence. It has previously been noted that such placement of anRBS results in robust translation in plant chloroplasts (see U.S.Application 2004/0014174, incorporated herein by reference), and thatpolypeptides that an advantage of expressing polypeptides inchloroplasts is that the polypeptides do not proceed through cellularcompartments typically traversed by polypeptides expressed from anuclear gene and, therefore, are not subject to certainpost-translational modifications such as glycosylation. As such, thepolypeptides and protein complexes produced by some methods of theinvention can be expected to be produced without such post-translationalmodification.

The following discussion is provided by way of background only andapplicant does not intend the disclosed invention to be limited, eitherin scope, or by theory, to the disclosure of mechanisms of chloroplastgene regulation. In chloroplasts, ribosome binding and propertranslation start site selection are thought to be mediated, at least inpart, by cis-acting RNA elements. One example of a potential regulatorhas been identified within the 5′UTR's of chloroplast mRNAs (Alexanderet al., Nucl. Acids Res. 26:2265-2272, 1998; Hirose and Sugiura, EMBO J.15:1687-1695, 1996; Mayfield et al., J. Cell Biol. 127:1537-1545, 1994;Sakamoto et al., Plant J. 6:503-512, 1994, each of which is incorporatedherein by reference). These elements may interact with nuclear-encodedfactors.

Many chloroplast mRNAs contain elements resembling prokaryotic RBSelements (Bonham-Smith and Bourque, Nucl. Acids Res. 17:2057-2080, 1989;Ruf and Kossel, FEBS Lett. 240:41-44, 1988, each of which isincorporated herein by reference). However, the functional utility ofthese RBS sequences in chloroplast translation has been unclear asseveral studies have shown differing effects of these elements ontranslation (Betts and Spremulli, J. Biol. Chem. 269:26456-26465, 1994;Hirose et al., FEBS Lett. 430:257-260, 1998; Fargo et al., Mol. Gen.Genet. 257:271-282, 1998; Koo and Spremulli, J. Biol. Chem.269:7494-7500, 1994; Rochaix, Plant Mol. Biol. 32:327-341, 1996).Interpretation of these results has been complicated by the lack of aconsensus for chloroplast RBS elements, and because the mutationsgenerated to study these putative RBS sequences may have altered thecontext of other important sequences within the 5′UTR.

Some aspects (e.g., vectors) of the present invention may include anRBS. Such RBSs can be chemically synthesized, or can be isolated from anaturally occurring nucleic acid molecule (e.g., isolation from achloroplast gene). In addition, to an RBS, embodiments with a 5′UTR caninclude transcriptional regulatory elements such as a promoter. As withRBSs utilized for the present invention, a 5′UTR may be chemicallysynthesized, or can be isolated from a naturally occurring nucleic acidmolecule. Non-limiting examples of 5′UTRs which may be used for thepresent invention include, but art not limited to, an atpA 5′UTR; a psbC5′UTR, a psbD 5′UTR, a psbA 5′UTR, a rbcL 5′UTR and/or a 16S rRNA 5′UTR.A ribonucleotide sequence may further include an initiation codon,(e.g., an AUG codon), operatively linked to an RBS. Initiation codonsmay be endogenous (e.g., naturally occurring in a cloned gene) or can besynthetic (e.g., inserted in a linker polypeptide or PCR primer).

An isolated ribonucleotide sequence may be obtained by any method knownin the art, including, but not limited to being chemically synthesized,generated using an enzymatic method, (e.g., generated from a DNA or RNAtemplate using a DNA dependent RNA polymerase or an RNA dependent RNApolymerase). A DNA template encoding the ribonucleotide of the inventioncan be chemically synthesized, can be isolated from a naturallyoccurring DNA molecule, or can be derived from a naturally occurring DNAsequence that is modified to have the required characteristics.

The term “polynucleotide” or “nucleotide sequence” or “nucleic acidmolecule” is used broadly herein to mean a sequence of two or moredeoxyribonucleotides or ribonucleotides that are linked together by aphosphodiester bond. As such, the terms include RNA and DNA, which canbe a gene or a portion thereof, a cDNA, a synthetic polydeoxyribonucleicacid sequence, or the like, and can be single stranded or doublestranded, as well as a DNA/RNA hybrid. Furthermore, the terms as usedherein include naturally occurring nucleic acid molecules, which can beisolated from a cell, as well as synthetic polynucleotides, which can beprepared, for example, by methods of chemical synthesis or by enzymaticmethods such as by the polymerase chain reaction (PCR). It should berecognized that the different terms are used only for convenience ofdiscussion so as to distinguish, for example, different components of acomposition, except that the term “synthetic polynucleotide” as usedherein refers to a polynucleotide that has been modified to reflectchloroplast codon usage.

In general, the nucleotides comprising a polynucleotide are naturallyoccurring deoxyribonucleotides, such as adenine, cytosine, guanine orthymine linked to 2′-deoxyribose, or ribonucleotides such as adenine,cytosine, guanine or uracil linked to ribose. Depending on the use,however, a polynucleotide also can contain nucleotide analogs, includingnon-naturally occurring synthetic nucleotides or modified naturallyoccurring nucleotides. Nucleotide analogs are well known in the art andcommercially available, as are polynucleotides containing suchnucleotide analogs (Lin et al., Nucl. Acids Res. 22:5220-5234, 1994;Jellinek et al., Biochemistry 34:11363-11372, 1995; Pagratis et al.,Nature Biotechnol. 15:68-73, 1997). Generally, a phosphodiester bondlinks the nucleotides of a polynucleotide of the present invention,however other bonds, including a thiodiester bond, a phosphorothioatebond, a peptide-like bond and any other bond known in the art may beutilized to produce synthetic polynucleotides (Tam et al., Nucl. AcidsRes. 22:977-986, 1994; Ecker and Crooke, BioTechnology 13:351360, 1995).

A polynucleotide comprising naturally occurring nucleotides andphosphodiester bonds can be chemically synthesized or can be producedusing recombinant DNA methods, using an appropriate polynucleotide as atemplate. In comparison, a polynucleotide comprising nucleotide analogsor covalent bonds other than phosphodiester bonds generally arechemically synthesized, although an enzyme such as T7 polymerase canincorporate certain types of nucleotide analogs into a polynucleotideand, therefore, can be used to produce such a polynucleotiderecombinantly from an appropriate template (Jellinek et al., supra,1995). Polynucleotides useful for practicing a method of the presentinvention may be isolated from any organism. Typically, thebiodegradative enzymes utilized in practicing the present invention areencoded by nucleotide sequences from bacteria or fungi. Non-limitingexamples of such enzymes and their sources are shown in Table I. Suchpolynucleotides may be isolated and/or synthesized by any means known inthe art, including, but not limited to cloning, sub-cloning, and PCR.

One or more codons of an encoding polynucleotide can be biased toreflect chloroplast codon usage. Most amino acids are encoded by two ormore different (degenerate) codons, and it is well recognized thatvarious organisms utilize certain codons in preference to others. Suchpreferential codon usage, which also is utilized in chloroplasts, isreferred to herein as “chloroplast codon usage”. The codon bias ofChlamydomonas reinihardtii has been reported. See U.S. Application2004/0014174.

The term “biased,” when used in reference to a codon, means that thesequence of a codon in a polynucleotide has been changed such that thecodon is one that is used preferentially in the target which the bias isfor, e.g., alga cells, chloroplasts, or the like. A polynucleotide thatis biased for chloroplast codon usage can be synthesized de novo, or canbe genetically modified using routine recombinant DNA techniques, forexample, by a site directed mutagenesis method, to change one or morecodons such that they are biased for chloroplast codon usage.Chloroplast codon bias can be variously skewed in different plants,including, for example, in alga chloroplasts as compared to tobacco.Generally, the chloroplast codon bias selected reflects chloroplastcodon usage of the plant which is being transformed with the nucleicacids of the present invention. For example, where C. reinhardtii is thehost, the chloroplast codon usage is biased to reflect alga chloroplastcodon usage (about 74.6% AT bias in the third codon position).

One method of the invention can be performed using a polynucleotide thatencodes a first polypeptide and at least a second polypeptide. As such,the polynucleotide can encode, for example, a first polypeptide and asecond polypeptide; a first polypeptide, a second polypeptide, and athird polypeptide; etc. Furthermore, any or all of the encodedpolypeptides can be the same or different. The polypeptides expressed inchloroplasts of the microalga C. reinhardtii may be assembled to formfunctional polypeptides and protein complexes. As such, a method of theinvention provides a means to produce functional protein complexes,including, for example, dimers, trimers, and tetramers, wherein thesubunits of the complexes can be the same or different (e.g., homodimersor heterodimers, respectively).

The term “recombinant nucleic acid molecule” is used herein to refer toa polynucleotide that is manipulated by human intervention. Arecombinant nucleic acid molecule can contain two or more nucleotidesequences that are linked in a manner such that the product is not foundin a cell in nature. In particular, the two or more nucleotide sequencescan be operatively linked and, for example, can encode a fusionpolypeptide, or can comprise an encoding nucleotide sequence and aregulatory element. A recombinant nucleic acid molecule also can bebased on, but manipulated so as to be different, from a naturallyoccurring polynucleotide, (e.g. biased for chloroplast codon usage,insertion of a restriction enzyme site, insertion of a promoter,insertion of an origin of replication). A recombinant nucleic acidmolecule may further contain a peptide tag (e.g., His-6 tag), which canfacilitate identification of expression of the polypeptide in a cell.Additional tags include, for example: a FLAG epitope, a c-myc epitope;biotin; and glutathione S-transferase. Such tags can be detected by anymethod known in the art (e.g., anti-tag antibodies, streptavidin). Suchtags may also be used to isolate the operatively linked polypeptide(s),for example by affinity chromatography.

Composition:

Nucleic Acids

The compositions herein comprise nucleic acids which encode one or moredifferent biomass degrading enzymes and/or one or more differentbiomass-production modulating agent and vectors of such nucleic acids.The nucleic acids can be heterologous to a photosynthetic host cell towhich they are inserted. The vector can include one or a plurality ofcopies of the nucleic acids which encode the biomass degrading enzymesand/or one or a plurality of copies of the nucleic acids which encodethe biomass-production modulating agents. When using a plurality ofcopies, at least 2, 3, 4, 5, 6 7, 8, 9, or 10 copies of the nucleicacids (e.g., encoding a single biomass degrading enzyme) can be insertedinto a single vector. This allows for an increased level of theirproduction in the host cell.

A recombinant nucleic acid molecule useful in a method of the inventioncan be contained in a vector. Furthermore, where the method is performedusing a second (or more) recombinant nucleic acid molecules, the secondrecombinant nucleic acid molecule also can be contained in a vector,which can, but need not, be the same vector as that containing the firstrecombinant nucleic acid molecule. The vector can be any vector usefulfor introducing a polynucleotide into a chloroplast and, preferably,includes a nucleotide sequence of chloroplast genomic DNA that issufficient to undergo homologous recombination with chloroplast genomicDNA, for example, a nucleotide sequence comprising about 400 to 1500 ormore substantially contiguous nucleotides of chloroplast genomic DNA.Chloroplast vectors and methods for selecting regions of a chloroplastgenome for use as a vector are well known (see, for example, Bock, J.Mol. Biol. 312:425-438, 2001; see, also, Staub and Maliga, Plant Cell4:39-45, 1992; Kavanagh et al., Genetics 152:1111-1122, 1999, each ofwhich is incorporated herein by reference).

In some instances, such vectors include promoters. Promoters useful forthe present invention may come from any source (e.g., viral, bacterial,fungal, protist, animal). The promoters contemplated herein can bespecific to photosynthetic organisms, non-vascular photosyntheticorganisms, and vascular photosynthetic organisms (e.g., algae, floweringplants). As used herein, the term “non-vascular photosyntheticorganism,” refers to any macroscopic or microscopic organism, including,but not limited to, algae, cyanobacteria and photosynthetic bacteria,which does not have a vascular system such as that found in higherplants. In some instances, the nucleic acids above are inserted into avector that comprises a promoter of a photosynthetic organism, e.g.,algae. The promoter can be a promoter for expression in a chloroplastand/or other plastid. In some instances, the nucleic acids arechloroplast based. Examples of promoters contemplated for insertion ofany of the nucleic acids herein into the chloroplast include thosedisclosed in US Application No. 2004/0014174. The promoter can be aconstitutive promoter or an inducible promoter. A promoter typicallyincludes necessary nucleic acid sequences near the start site oftranscription, (e.g., a TATA element).

A “constitutive” promoter is a promoter that is active under mostenvironmental and developmental conditions. An “inducible” promoter is apromoter that is active under environmental or developmental regulation.Examples of inducible promoters/regulatory elements include, forexample, a nitrate-inducible promoter (Back et al, Plant Mol. Biol. 17:9(1991)), or a light-inducible promoter, (Feinbaum et al, MoI Gen. Genet.226:449 (1991); Lam and Chua, Science 248:471 (1990)), or a heatresponsive promoter (Muller et al., Gene 111: 165-73 (1992)).

The entire chloroplast genome of C. reinhardtii is available to thepublic on the world wide web, at the URL“biology.duke.edu/chlamy_genome/-chloro.html” (see “view complete genomeas text file” link and “maps of the chloroplast genome” link), each ofwhich is incorporated herein by reference (J. Maul, J. W. Lilly, and D.B. Stern, unpublished results; revised Jan. 28, 2002; to be published asGenBank Acc. No. AF396929). Generally, the nucleotide sequence of thechloroplast genomic DNA is selected such that it is not a portion of agene, including a regulatory sequence or coding sequence, particularly agene that, if disrupted due to the homologous recombination event, wouldproduce a deleterious effect with respect to the chloroplast, forexample, for replication of the chloroplast genome, or to a plant cellcontaining the chloroplast. In this respect, the website containing theC. reinhardtii chloroplast genome sequence also provides maps showingcoding and non-coding regions of the chloroplast genome, thusfacilitating selection of a sequence useful for constructing a vector ofthe invention. For example, the chloroplast vector, p322, which was usedin experiments disclosed herein, is a clone extending from the Eco (EcoRI) site at about position 143.1 kb to the Xho (Xho I) site at aboutposition 148.5 kb (see, world wide web, at the URL“biology.duke.edu/chlamy_genome/chloro.html”, and clicking on “maps ofthe chloroplast genome” link, and “140-150 kb” link; also accessibledirectly on world wide web at URL“biology.duke.edu/chlam-y/chloro/chlorol40.html”; see, also, Example 1).

A vector utilized in the practice of the invention also can contain oneor more additional nucleotide sequences that confer desirablecharacteristics on the vector, including, for example, sequences such ascloning sites that facilitate manipulation of the vector, regulatoryelements that direct replication of the vector or transcription ofnucleotide sequences contain therein, sequences that encode a selectablemarker, and the like. As such, the vector can contain, for example, oneor more cloning sites such as a multiple cloning site, which can, butneed not, be positioned such that a heterologous polynucleotide can beinserted into the vector and operatively linked to a desired element.The vector also can contain a prokaryote origin of replication (ori),for example, an E. coli ori or a cosmid ori, thus allowing passage ofthe vector in a prokaryote host cell, as well as in a plant chloroplast,as desired.

A regulatory element, as the term is used herein, broadly refers to anucleotide sequence that regulates the transcription or translation of apolynucleotide or the localization of a polypeptide to which it isoperatively linked. Examples include, but are not limited to, an RBS, apromoter, enhancer, transcription terminator, an initiation (start)codon, a splicing signal for intron excision and maintenance of acorrect reading frame, a STOP codon, an amber or ochre codon, an IRES.Additionally, a cell compartmentalization signal (i.e., a sequence thattargets a polypeptide to the cytosol, nucleus, chloroplast membrane orcell membrane). Such signals are well known in the art and have beenwidely reported (see, e.g., U.S. Pat. No. 5,776,689).

A vector or other recombinant nucleic acid molecule may include anucleotide sequence encoding a reporter polypeptide or other selectablemarker. The term “reporter” or “selectable marker” refers to apolynucleotide (or encoded polypeptide) that confers a detectablephenotype. A reporter generally encodes a detectable polypeptide, forexample, a green fluorescent protein or an enzyme such as luciferase,which, when contacted with an appropriate agent (a particular wavelengthof light or luciferin, respectively) generates a signal that can bedetected by eye or using appropriate instrumentation (Giacomin, PlantSci. 116:59-72, 1996; Scikantha, J. Bacteriol. 178:121, 1996; Gerdes,FEBS Lett. 389:44-47, 1996; see, also, Jefferson, EMBO J. 6:3901-3907,1997, fl-glucuronidase). A selectable marker generally is a moleculethat, when present or expressed in a cell, provides a selectiveadvantage (or disadvantage) to the cell containing the marker, forexample, the ability to grow in the presence of an agent that otherwisewould kill the cell.

A selectable marker can provide a means to obtain prokaryotic cells orplant cells or both that express the marker and, therefore, can beuseful as a component of a vector of the invention (see, for example,Bock, supra, 2001). Examples of selectable markers include, but are notlimited to, those that confer antimetabolite resistance, for example,dihydrofolate reductase, which confers resistance to methotrexate(Reiss, Plant Physiol. (Life Sci. Adv.) 13:143-149, 1994); neomycinphosphotransferase, which confers resistance to the aminoglycosidesneomycin, kanamycin and paromycin (Herrera-Estrella, EMBO J. 2:987-995,1983), hygro, which confers resistance to hygromycin (Marsh, Gene32:481-485, 1984), trpB, which allows cells to utilize indole in placeof tryptophan; hisD, which allows cells to utilize histinol in place ofhistidine (Hartman, Proc. Natl. Acad. Sci., USA 85:8047, 1988);mannose-6-phosphate isomerase which allows cells to utilize mannose (WO94/20627); ornithine decarboxylase, which confers resistance to theornithine decarboxylase inhibitor, 2-(difluoromethyl)-DL-ornithine(DFMO; McConlogue, 1987, In: Current Communications in MolecularBiology, Cold Spring Harbor Laboratory ed.); and deaminase fromAspergillus terreus, which confers resistance to Blasticidin S (Tamura,Biosci. Biotechnol. Biochem. 59:2336-2338, 1995). Additional selectablemarkers include those that confer herbicide resistance, for example,phosphinothricin acetyltransferase gene, which confers resistance tophosphinothricin (White et al., Nucl. Acids Res. 18:1062, 1990; Spenceret al., Theor. Appl. Genet. 79:625-631, 1990), a mutant EPSPV-synthase,which confers glyphosate resistance (Hinchee et al., BioTechnology91:915-922, 1998), a mutant acetolactate synthase, which confersimidazolone or sulfonylurea resistance (Lee et al., EMBO J. 7:1241-1248,1988), a mutant psbA, which confers resistance to atrazine (Smeda etal., Plant Physiol. 103:911-917, 1993), or a mutant protoporphyrinogenoxidase (see U.S. Pat. No. 5,767,373), or other markers conferringresistance to an herbicide such as glufosinate. Selectable markersinclude polynucleotides that confer dihydrofolate reductase (DHFR) orneomycin resistance for eukaryotic cells and tetracycline; ampicillinresistance for prokaryotes such as E. coli; and bleomycin, gentamycin,glyphosate, hygromycin, kanamycin, methotrexate, phleomycin,phosphinotricin, spectinomycin, streptomycin, sulfonamide andsulfonylurea resistance in plants (see, for example, Maliga et al.,Methods in Plant Molecular Biology, Cold Spring Harbor Laboratory Press,1995, page 39).

Reporter genes have been successfully used in chloroplasts of higherplants, and high levels of recombinant protein expression have beenreported. In addition, reporter genes have been used in the chloroplastof C. reinhardtii, but, in most cases very low amounts of protein wereproduced. Reporter genes greatly enhance the ability to monitor geneexpression in a number of biological organisms. In chloroplasts ofhigher plants, β-glucuronidase (uidA, Staub and Maliga, EMBO J.12:601-606, 1993), neomycin phosphotransferase (nptII, Carrer et al.,Mol. Gen. Genet. 241:49-56, 1993), adenosyl-3-adenyltransf-erase (aadA,Svab and Maliga, Proc. Natl. Acad. Sci., USA 90:913-917, 1993), and theAequorea victoria GFP (Sidorov et al., Plant J. 19:209-216, 1999) havebeen used as reporter genes (Heifetz, Biochemie 82:655-666, 2000). Eachof these genes has attributes that make them useful reporters ofchloroplast gene expression, such as ease of analysis, sensitivity, orthe ability to examine expression in situ. Based upon these studies,other heterologous proteins have been expressed in the chloroplasts ofhigher plants such as Bacillus thuringiensis Cry toxins, conferringresistance to insect herbivores (Kota et al., Proc. Natl. Acad. Sci.,USA 96:1840-1845, 1999), or human somatotropin (Staub et al., Nat.Biotechnol. 18:333-338, 2000), a potential biopharmaceutical. Severalreporter genes have been expressed in the chloroplast of the eukaryoticgreen alga, C. reinhardtii, including aadA (Goldschmidt-Clermont, Nucl.Acids Res. 19:4083-4089 1991; Zerges and Rochaix, Mol. Cell. Biol.14:5268-5277, 1994), uidA (Sakamoto et al., Proc. Natl. Acad. Sci., USA90:477-501, 19933, Ishikura et al., J. Biosci. Bioeng. 87:307-314 1999),Renilla luciferase (Minko et al., Mol. Gen. Genet. 262:421-425, 1999)and the amino glycoside phosphotransferase from Acinetobacter baumanii,aphA6 (Bateman and Purton, Mol. Gen. Genet. 263:404-410, 2000).

In some instances, the vectors of the present invention will containelements such as an E. coli or S. cerevisiae origin of replication. Suchfeatures, combined with appropriate selectable markers, allows for thevector to be “shuttled” between the target host cell and the bacterialand/or yeast cell. The ability to passage a shuttle vector of theinvention in a secondary host may allow for more convenient manipulationof the features of the vector. For example, a reaction mixturecontaining the vector and putative inserted polynucleotides of interestcan be transformed into prokaryote host cells such as E. coli, amplifiedand collected using routine methods, and examined to identify vectorscontaining an insert or construct of interest. If desired, the vectorcan be further manipulated, for example, by performing site directedmutagenesis of the inserted polynucleotide, then again amplifying andselecting vectors having a mutated polynucleotide of interest. A shuttlevector then can be introduced into plant cell chloroplasts, wherein apolypeptide of interest can be expressed and, if desired, isolatedaccording to a method of the invention.

A polynucleotide or recombinant nucleic acid molecule of the invention,can be introduced into plant chloroplasts using any method known in theart. A polynucleotide can be introduced into a cell by a variety ofmethods, which are well known in the art and selected, in part, based onthe particular host cell. For example, the polynucleotide can beintroduced into a plant cell using a direct gene transfer method such aselectroporation or microprojectile mediated (biolistic) transformationusing a particle gun, or the “glass bead method,” or by pollen-mediatedtransformation, liposome-mediated transformation, transformation usingwounded or enzyme-degraded immature embryos, or wounded orenzyme-degraded embryogenic callus (Potrykus, Ann. Rev. Plant. Physiol.Plant Mol. Biol. 42:205-225, 1991).

The term “exogenous” is used herein in a comparative sense to indicatethat a nucleotide sequence (or polypeptide) being referred to is from asource other than a reference source, or is linked to a secondnucleotide sequence (or polypeptide) with which it is not normallyassociated, or is modified such that it is in a form that is notnormally associated with a reference material. For example, apolynucleotide encoding an biomass degrading enzyme is heterologous withrespect to a nucleotide sequence of a plant chloroplast, as are thecomponents of a recombinant nucleic acid molecule comprising, forexample, a first nucleotide sequence operatively linked to a secondnucleotide sequence, as is a mutated polynucleotide introduced into achloroplast where the mutant polynucleotide is not normally found in thechloroplast.

Plastid transformation is a routine and well known method forintroducing a polynucleotide into a plant cell chloroplast (see U.S.Pat. Nos. 5,451,513, 5,545,817, and 5,545,818; WO 95/16783; McBride etal., Proc. Natl. Acad. Sci., USA 91:7301-7305, 1994). In someembodiments, chloroplast transformation involves introducing regions ofchloroplast DNA flanking a desired nucleotide sequence, allowing forhomologous recombination of the exogenous DNA into the targetchloroplast genome. In some instances one to 1.5 kb flanking nucleotidesequences of chloroplast genomic DNA may be used. Using this method,point mutations in the chloroplast 16S rRNA and rps12 genes, whichconfer resistance to spectinomycin and streptomycin, can be utilized asselectable markers for transformation (Svab et al., Proc. Natl. Acad.Sci., USA 87:8526-8530, 1990), and can result in stable homoplasmictransformants, at a frequency of approximately one per 100 bombardmentsof target leaves.

Microprojectile mediated transformation also can be used to introduce apolynucleotide into a plant cell chloroplast (Klein et al., Nature327:70-73, 1987). This method utilizes microprojectiles such as gold ortungsten, which are coated with the desired polynucleotide byprecipitation with calcium chloride, spermidine or polyethylene glycol.The microprojectile particles are accelerated at high speed into a planttissue using a device such as the BIOLISTIC PD-1000 particle gun(BioRad; Hercules Calif.). Methods for the transformation usingbiolistic methods are well known in the art (see, e.g.; Christou, Trendsin Plant Science 1:423-431, 1996). Microprojectile mediatedtransformation has been used, for example, to generate a variety oftransgenic plant species, including cotton, tobacco, corn, hybrid poplarand papaya. Important cereal crops such as wheat, oat, barley, sorghumand rice also have been transformed using microprojectile mediateddelivery (Duan et al., Nature Biotech. 14:494-498, 1996; Shimamoto,Curr. Opin. Biotech. 5:158-162, 1994). The transformation of mostdicotyledonous plants is possible with the methods described above.Transformation of monocotyledonous plants also can be transformed using,for example, biolistic methods as described above, protoplasttransformation, electroporation of partially permeabilized cells,introduction of DNA using glass fibers, the glass bead agitation method,and the like.

Transformation frequency may be increased by replacement of recessiverRNA or r-protein antibiotic resistance genes with a dominant selectablemarker, including, but not limited to the bacterial aadA gene (Svab andMaliga, Proc. Natl. Acad. Sci., USA 90:913-917, 1993). Approximately 15to 20 cell division cycles following transformation are generallyrequired to reach a homoplastidic state. It is apparent to one of skillin the art that a chloroplast may contain multiple copies of its genome,and therefore, the term “homoplasmic” or “homoplasmy” refers to thestate where all copies of a particular locus of interest aresubstantially identical. Plastid expression, in which genes are insertedby homologous recombination into all of the several thousand copies ofthe circular plastid genome present in each plant cell, takes advantageof the enormous copy number advantage over nuclear-expressed genes topermit expression levels that can readily exceed 10% of the totalsoluble plant protein.

The methods of the present invention are exemplified using themicroalga, C. reinhardtii. The use of microalgae to express apolypeptide or protein complex according to a method of the inventionprovides the advantage that large populations of the microalgae can begrown, including commercially (Cyanotech Corp.; Kailua-Kona Hi.), thusallowing for production and, if desired, isolation of large amounts of adesired product. However, the ability to express, for example,functional mammalian polypeptides, including protein complexes, in thechloroplasts of any plant allows for production of crops of such plantsand, therefore, the ability to conveniently produce large amounts of thepolypeptides. Accordingly, the methods of the invention can be practicedusing any plant having chloroplasts, including, for example, macroalgae,for example, marine algae and seaweeds, as well as plants that grow insoil, for example, corn (Zea mays), Brassica sp. (e.g., B. napus, B.rapa, B. juncea), particularly those Brassica species useful as sourcesof seed oil, alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secalecereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet (e.g.,pearl millet (Pennisetum glaucum), proso millet (Panicum miliaceum),foxtail millet (Setaria italica), finger millet (Eleusine coracana)),sunflower (Helianthus annuus), safflower (Carthamus tinctorius), wheat(Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum),potato (Solanum tuberosum), peanuts (Arachis hypogaea), cotton(Gossypium barbadense, Gossypium hirsutum), sweet potato (Ipomoeabatatus), cassaya (Manihot esculenta), coffee (Cofea spp.), coconut(Cocos nucifera), pineapple (Ananas comosus), citrus trees (Citrusspp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musaspp.), avocado (Persea ultilane), fig (Ficus casica), guava (Psidiumguajava), mango (Mangifera indica), olive (Olea europaea), papaya(Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamiaintegrifolia), almond (Prunus amygdalus), sugar beets (Beta vulgaris),sugar cane (Saccharum spp.), oats, duckweed (Lemna), barley, tomatoes(Lycopersicon esculentum), lettuce (e.g., Lactuca sativa), green beans(Phaseolus vulgaris), lima beans (Phaseolus limensis), peas (Lathyrusspp.), and members of the genus Cucumis such as cucumber (C. sativus),cantaloupe (C. cantalupensis), and musk melon (C. melo). Ornamentalssuch as azalea (Rhododendron spp.), hydrangea (Macrophylla hydrangea),hibiscus (Hibiscus rosasanensis), roses (Rosa spp.), tulips (Tulipaspp.), daffodils (Narcissus spp.), petunias (Petunia hybrida), carnation(Dianthus caryophyllus), poinsettia (Euphorbia pulcherrima), andchrysanthemum are also included. Additional ornamentals useful forpracticing a method of the invention include impatiens, Begonia,Pelargonium, Viola, Cyclamen, Verbena, Vinca, Tagetes, Primula, SaintPaulia, Agertum, Amaranthus, Antihirrhinum, Aquilegia, Cineraria,Clover, Cosmo, Cowpea, Dahlia, Datura, Delphinium, Gerbera, Gladiolus,Gloxinia, Hippeastrum, Mesembryanthemum, Salpiglossos, and Zinnia.Conifers that may be employed in practicing the present inventioninclude, for example, pines such as loblolly pine (Pinus taeda), slashpine (Pinus elliotii), ponderosa pine (Pinus ponderosa), lodgepole pine(Pinus contorta), and Monterey pine (Pinus radiata), Douglas-fir(Pseudotsuga menziesii); Western hemlock (Tsuga ultilane); Sitka spruce(Picea glauca); redwood (Sequoia seinpervirens); true firs such assilver fir (Abies amabilis) and balsam fir (Abies balsamea); and cedarssuch as Western red cedar (Thuja plicata) and Alaska yellow-cedar(Chamaecyparis nootkatensis).

Leguminous plants useful for practicing a method of the inventioninclude beans and peas. Beans include guar, locust bean, fenugreek,soybean, garden beans, cowpea, mung bean, lima bean, fava bean, lentils,chickpea, etc. Legumes include, but are not limited to, Arachis, e.g.,peanuts, Vicia, e.g., crown vetch, hairy vetch, adzuki bean, mung bean,and chickpea, Lupinus, e.g., lupine, trifolium, Phaseolus, e.g., commonbean and lima bean, Pisum, e.g., field bean, Melilotus, e.g., clover,Medicago, e.g., alfalfa, Lotus, e.g., trefoil, lens, e.g., lentil, andfalse indigo. Preferred forage and turf grass for use in the methods ofthe invention include alfalfa, orchard grass, tall fescue, perennialryegrass, creeping bent grass, and redtop. Other plants useful in theinvention include Acacia, aneth, artichoke, arugula, blackberry, canola,cilantro, clementines, escarole, eucalyptus, fennel, grapefruit, honeydew, jicama, kiwifruit, lemon, lime, mushroom, nut, okra, orange,parsley, persimmon, plantain, pomegranate, poplar, radiata pine,radicchio, Southern pine, sweetgum, tangerine, triticale, vine, yams,apple, pear, quince, cherry, apricot, melon, hemp, buckwheat, grape,raspberry, chenopodium, blueberry, nectarine, peach, plum, strawberry,watermelon, eggplant, pepper, cauliflower, Brassica, e.g., broccoli,cabbage, ultilan sprouts, onion, carrot, leek, beet, broad bean, celery,radish, pumpkin, endive, gourd, garlic, snapbean, spinach, squash,turnip, ultilane, chicory, groundnut and zucchini. Thus, thecompositions contemplated herein include host organisms comprising anyof the above nucleic acids. The host organism can be anychloroplast-containing organism.

The term “plant” is used broadly herein to refer to a eukaryoticorganism containing plastids, particularly chloroplasts, and includesany such organism at any stage of development, or to part of a plant,including a plant cutting, a plant cell, a plant cell culture, a plantorgan, a plant seed, and a plantlet. A plant cell is the structural andphysiological unit of the plant, comprising a protoplast and a cellwall. A plant cell can be in the form of an isolated single cell or acultured cell, or can be part of higher organized unit, for example, aplant tissue, plant organ, or plant. Thus, a plant cell can be aprotoplast, a gamete producing cell, or a cell or collection of cellsthat can regenerate into a whole plant. As such, a seed, which comprisesmultiple plant cells and is capable of regenerating into a whole plant,is considered plant cell for purposes of this disclosure. A plant tissueor plant organ can be a seed, protoplast, callus, or any other groups ofplant cells that is organized into a structural or functional unit.Particularly useful parts of a plant include harvestable parts and partsuseful for propagation of progeny plants. A harvestable part of a plantcan be any useful part of a plant, for example, flowers, pollen,seedlings, tubers, leaves, stems, fruit, seeds, roots, and the like. Apart of a plant useful for propagation includes, for example, seeds,fruits, cuttings, seedlings, tubers, rootstocks, and the like.

A method of the invention can generate a plant containing chloroplaststhat are genetically modified to contain a stably integratedpolynucleotide (Hager and Bock, Appl. Microbiol. Biotechnol. 54:302-310,2000). Accordingly, the present invention further provides a transgenic(transplastomic) plant, e.g. C. reinhardtii, which comprises one or morechloroplasts containing a polynucleotide encoding one or moreheterologous polypeptides, including polypeptides that can specificallyassociate to form a functional protein complex.

In some instances, transformants and/or transplastomic plants comprisinga recombinant polynucleotide encoding a single enzyme of a particularbiodegradative pathway (e.g., the cellulosic pathway), may be combinedwith transformants comprising recombinant polynucleotides encoding theother enzymes of the biodegradative pathway. For example, where abiochemical pathway utilizes four enzymes to produce a product from asubstrate, four transformant lines may be combined to provide theenzymes of that pathway. Such combinations may contain as manytransformant lines as is necessary to comprise a mixture of cellsproducing the entire enzyme pathway, or a portion thereof. Additionally,such combinations may comprise different ratios of cells of thedifferent transformants. For example, where one enzyme of a degradativepathway is the rate limiting step in the pathway, a combination of cellsmay contain 2, 3, 4, 5, 6, 7, 8, 9, 10 times or higher numbers of cellsproducing the rate limiting enzyme. One of skill in the art willrecognize that multiple combinations of ratios of transformants may beachieved through simple methods (e.g., weighing dried tranformants andcombining). Alternately, individual enzymes may be isolated from thetransformants (e.g., “cracking” algal transformants to isolatesequestered enzymes) and then combined following isolation. Suchapproaches may allow for tailoring of enzyme concentrations to differentbiomass or other substrate materials which may contain differentrelative ratios of substrates or other components.

In some instances, a protein produced by a transgenic organism of thepresent invention is isolated after it is produced. Therefore, thepresent invention also contemplates a method of producing a heterologouspolypeptide or protein complex in a chloroplast or in a transgenic plantwhich may include a step of isolating an expressed polypeptide orprotein complex from the plant cell chloroplasts. As used herein, theterm “isolated” or “substantially purified” means that a polypeptide orpolynucleotide being referred to is in a form that is relatively free ofproteins, nucleic acids, lipids, carbohydrates or other materials withwhich it is naturally associated. An isolated polypeptide (orpolynucleotide) may constitute at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 6061, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or100 percent of a sample.

A polypeptide or protein complex can be isolated from chloroplasts usingany method suitable for the particular polypeptide or protein complex,including, for example, salt fractionation methods and chromatographymethods such as an affinity chromatography method using a ligand orreceptor that specifically binds the polypeptide or protein complex. Adetermination that a polypeptide or protein complex produced accordingto a method of the invention is in an isolated form can be made usingwell known methods, for example, by performing electrophoresis andidentifying the particular molecule as a relatively discrete band or theparticular complex as one of a series of bands. Accordingly, the presentinvention also provides an isolated polypeptide or protein complexproduced by a method of the invention. In some instances, an enzyme ofthe present invention may be produced but sequestered in thechloroplast. In such embodiments, access to the active enzyme may be hadupon “cracking” the cells containing the enzyme (e.g., using mechanical,chemical, and/or biological means to disrupt the cell wall). The timingof such cracking may be planned to occur at the time the enzyme(s)produced by the cells are to be utilized to perform their enzymaticcapabilities. In other instances, the enzyme may be secreted by the hostcell. In such instances, the enzyme may be collected directly from theculture medium of the organism. Enzymes present in such media may beused directly, without purification, may be dried (e.g., air dried,lyophilized), and/or may be subjected to purification by any means knownin the art (e.g., affinity chromatography, high performance liquidchromatography).

Examples of biomass-degrading enzymes and the nucleic acids that encodethose enzymes are shown in Table I. Non-limiting examples ofbiomass-degrading enzymes include: cellulolytic enzymes,hemicellulolytic enzymes, pectinolytic enzymes, xylanases, ligninolyticenzymes, cellulases, cellobiases, softening enzymes (e.g.,endopolygalacturonase), amylases, lipases, proteases, RNAses, DNAses,inulinase, lysing enzymes, phospholipases, pectinase, pullulanase,glucose isomerase, endoxylanase, beta-xylosidase,alpha-L-arabinofuranosidase, alpha-glucoronidase, alpha-galactosidase,acetylxylan esterase, and feruloyl esterase. Examples of genes thatencode such enzymes include, but are not limited to, amylases,cellulases, hemicellulases, (e.g., β-glucosidase, endocellulase,exocellulase), exo-β-glucanase, endo-β-glucanase and xylanse(endoxylanase and exoxylanse). Examples of ligninolytic enzymes include,but are not limited to, lignin peroxidase and manganese peroxidase fromPhanerochaete chryososporium. One of skill in the art will recognizethat these enzymes are only a partial list of enzymes which could beused in the present invention.

TABLE 1 Examples of Biomass-degrading enzymes # Target (family) SourceAA NCBI prot. ID Exo-β-glucanase 1. CBH I (7) Trichoderma viride 514AAQ76092 2. CBH II (6) T. reesei 471 AAA72922 3. CBH I (7) Aspergillus540 BAA25183 aculeatus Endo-β-glucanase 4. EG I (7) T. reesei 459AAA34212 5. EG III (5) T. reesei 218 AAA34213 6. EG V (45) T. reesei 242CAA83846 7. EGL A (12) A. niger 239 CAA11964 β-glucosidase 8. BGL I (3)T. reesei 744 AAA18473 9. BGL II (1) T. reesei 466 BAA74959 10. BGL I(3) A. niger 860 ABG46337 Endoxylanase 11. XYN I (11) T. reesei 222CAA49293 12. XYN II (11) T. reesei 229 CAA49294

Biomass-production modulating agents include agents that increasebiomass production in an organism, e.g., photosynthetic organism.

Host Cells/Organism

The present invention also contemplates a host cell transformed with oneor more of the nucleic acids herein. In preferred embodiments, the hostcell is photosynthetic. In some cases, the host cell is photosyntheticand non-vascular. In other cases, the host cell is photosynthetic andvascular. The host cell can be eukaryotic or prokaryotic.

The host cell is transfected with a vector described herein (e.g., avector comprising one or more biomass degrading enzymes and/or one ormore biomass-production modulating agents). The vector may contain aplastid promoter or a nucleic promoter for transfecting a chloroplast orother plastid of the host cell. The vector may also encode a fusionprotein or agent that selectively targets the vector product to thechloroplast or other plastid. Transfection of a host cell can occurusing any method known in the art.

A host organism is an organism comprising a host cell. In preferredembodiments, the host organism is photosynthetic. A photosyntheticorganism is one that naturally photosynthesizes (has a plastid) or thatis genetically engineered or otherwise modified to be photosynthetic. Insome instances, a photosynthetic organism may be transformed with aconstruct of the invention which renders all or part of thephotosynthetic apparatus inoperable. In some instances it isnon-vascular and photosynthetic. The host cell can be prokaryotic.Examples of some prokaryotic organisms of the present invention include,but are not limited to cyanobacteria (e.g., Synechococcus,Synechocystis, Athrospira). The host organism can be unicellular ormulticellular. In most embodiments, the host organism is eukaryotic(e.g. green algae). Examples of organisms contemplated herein include,but are not limited to, rhodophyta, chlorophyta, heterokontophyta,tribophyta, glaucophyta, chlorarachniophytes, euglenoids, haptophyta,cryptomonads, dinoflagellata, and phytoplankton.

A host organism may be grown under conditions which permitphotosynthesis, however, this is not a requirement (e.g., a hostorganism may be grown in the absence of light). In some instances, thehost organism may be genetically modified in such a way thatphotosynthetic capability is diminished and/or destroyed (see examplesbelow). In growth conditions where a host organism is not capable ofphotosynthesis (e.g., because of the absence of light and/or geneticmodification), typically, the organism will be provided with thenecessary nutrients to support growth in the absence of photosynthesis.For example, a culture medium in (or on) which an organism is grown, maybe supplemented with any required nutrient, including an organic carbonsource, nitrogen source, phosphorous source, vitamins, metals, lipids,nucleic acids, micronutrients, or an organism-specific requirement.Organic carbon sources include any source of carbon which the hostorganism is able to metabolize including, but not limited to, acetate,simple carbohydrates (e.g., glucose, sucrose, lactose), complexcarbohydrates (e.g., starch, glycogen), proteins, and lipids. One ofskill in the art will recognize that not all organisms will be able tosufficiently metabolize a particular nutrient and that nutrient mixturesmay need to be modified from one organism to another in order to providethe appropriate nutrient mix.

A host organism can be grown on land, e.g., ponds, aqueducts, landfills,or in closed or partially closed bioreactor systems. The host organismsherein can also be grown directly in water, e.g., in ocean, sea, onlakes, rivers, reservoirs, etc. In embodiments where algae aremass-cultured, the algae can be grown in high density photobioreactorsMethods of mass-culturing algae are known. For example, algae can begrown in high density photobioreactors (see, e.g., Lee et al, Biotech.Bioengineering 44:1161-1167, 1994) and other bioreactors (such as thosefor sewage and waste water treatments) (e.g., Sawayama et al, Appl.Micro. Biotech., 41:729-731, 1994). Additionally, algae may bemass-cultured to remove heavy metals (e.g., Wilkinson, Biotech. Letters,11:861-864, 1989), hydrogen (e.g., U.S. Patent Application PublicationNo. 20030162273), and pharmaceutical compounds

In some cases, host organism(s) are grown near ethanol production plantsor other facilities or regions (e.g., electrical generating plants,concrete plants, oil refineries, other industrial facilities, cities,highways, etc.) generating CO₂. As such, the methods herein contemplatebusiness methods for selling carbon credits to ethanol plants or otherfacilities or regions generating CO₂ while making or catalyzing theproduction of fuels by growing one or more of the modified organismsdescribed herein near the ethanol production plant.

Biomass

As used herein, “biomass” is any organic material. In some instances,biomass is substantially free or free of starch and simple sugars.Biomass can be broken down into starch or simple sugars that can besubsequently utilized for the production of fuel. Any cellulosic orlignocellulosic material and materials comprising cellulose,hemicellulose, lignin, starch, oligosaccharides and/or monosaccharidesare also considered to be biomass. Biomass may also comprise additionalcomponents, such as protein and/or lipid. Biomass may be derived from asingle source, or biomass can comprise a mixture derived from more thanone source; for example, biomass could comprise a mixture of corn cobsand corn stover, or a mixture of grass and leaves. Biomass includes, butis not limited to, bioenergy crops, agricultural residues, municipalsolid waste, industrial solid waste, sludge from paper manufacture, yardwaste, wood and forestry waste. Examples of biomass include, but are notlimited to, corn grain, corn cobs, crop residues such as corn husks,corn stover, grasses, wheat, wheat straw, barley, barley straw, hay,rice straw, switchgrass, waste paper, sugar cane bagasse, sorghum, soy,components obtained from milling of grains, trees, branches, roots,leaves, wood chips, sawdust, paper, shrubs and bushes, vegetables,fruits, flowers and animal manure.

Agricultural waste is one form of biomass used for the production offuel. Non-limiting examples of agricultural waste include corn stover,wheat stover, and soy stover. Another source of biomass in thisinvention is a high cellulose content organism. A high cellulose contentorganism is an organism whose weight is at least 30% or moreattributable to biomass that is substantially free of starch or simplesugars. High cellulose content organism(s) can be selectively grown inlarge quantities to produce biomass, which can be degraded withbiomass-degrading enzyme(s) of this invention to create starch andsimple sugars. Examples of high cellulose content organisms include, butare not limited to: willow, duckweed, sugarbeets, and switchgrass.

A third example of biomass comprises organisms that are geneticallymodified to have an increased cellulose or biomass. Such organisms areoptionally photosynthetic and may comprise a host cell incorporating avector that encodes a biomass production-modulating agent. In someinstances, the same vector can encode both a biomassproduction-modulating agent and a biomass-degrading enzyme. In someinstances, the vector encoding the biomass production-modulating agentand the vector encoding the biomass degrading enzyme are separate.

Fuel Production

The present invention relates to methods of producing a biofuel. Suchmethods comprise expressing a gene encoding a biomass-degrading enzymein a photosynthetic organism (e.g., non-vascular). The method furthercomprises utilizing the biomass-degrading enzyme and breaking downbiomass with the enzyme. To produce a biofuel, the method may furtherinvolve refining the degraded biomass. The final product (e.g., ethanol)may then be mixed with one or more other biofuels.

The invention relates to a method of producing a biofuel comprisingexpressing a vector or vectors encoding a biomass-degrading enzyme, abiomass-degrading enzymatic pathway, and/or a biomassproduction-modulating agent in photosynthetic organism (e.g.,non-vascular). In this embodiment, the host cell comprising the vectorcould then both make and degrade its own biomass. The method cancomprise extracting only the product of the biomass degradation. In thismanner, the enzyme would not have to be extracted to use for thecreation of a biofuel. The production of the biofuel may further involverefining the product of the breaking down of the biomass. The productionof biofuel may also involve the utilization of saccharification tanks.Such devices are well known in the art, see, for example U.S. Pat. Nos.5,114,491; 5,534,075; and 5,559,031 (each of which is hereinincorporated by reference).

In some embodiments, the biofuel is ethanol or other biologicallyproduced alcohols. The refining may include a fermentation step toproduce ethanol from products of biomass degradation including starchand simple sugars. Thus, refining may include using microorganisms whichare capable of fermenting starch, simple sugars, and/or biomassmaterials, including, but not limited to Saccharomyces cerevisiae andZymomonas mobilis.

The following examples merely illustrate the invention disclosed herein,but do not limit it.

EXAMPLES Example 1 Production of Endo-β-glucanase in C. reinhardtii

In this example a nucleic acid encoding endo-β-glucanase from T. reeseiwas introduced into C. reinhardtii. Transforming DNA (SEQ ID NO. 20,Table 4) is shown graphically in FIG. 2A. In this instance the segmentlabeled “Transgene” is the endo-β-glucanase encoding gene (SEQ ID NO.16, Table 3), the segment labeled “psbA 5′UTR” is the 5′ UTR andpromoter sequence for the psbA gene from C. reinhardtii, the segmentlabeled “psbA 3′UTR” contains the 3′ UTR for the psbA gene from C.reinhardtii, and the segment labeled “Selection Marker” is the kanamycinresistance encoding gene from bacteria, which is regulated by the 5′ UTRand promoter sequence for the atpA gene from C. reinhardtii and the 3′UTR sequence for the rbcL gene from C. reinhardtii. The transgenecassette is targeted to the psbA loci of C. reinhardtii via the segmentslabeled “5′Homology” and “3′ Homology,” which are identical to sequencesof DNA flanking the psbA locus on the 5′ and 3′ sides, respectively. AllDNA manipulations carried out in the construction of this transformingDNA were essentially as described by Sambrook et al., Molecular Cloning:A Laboratory Manual (Cold Spring Harbor Laboratory Press 1989) and Cohenet al., Meth. Enzymol. 297, 192-208, 1998.

For these experiments, all transformations were carried out on C.reinhardtii strain 137c (mt+). Cells were grown to late log phase(approximately 7 days) in the presence of 0.5 mM 5-fluorodeoxyuridine inTAP medium (Gorman and Levine, Proc. Natl. Acad. Sci., USA 54:1665-1669,1965, which is incorporated herein by reference) at 23° C. underconstant illumination of 450 Lux on a rotary shaker set at 100 rpm.Fifty ml of cells were harvested by centrifugation at 4,000×g at 23° C.for 5 min. The supernatant was decanted and cells resuspended in 4 mlTAP medium for subsequent chloroplast transformation by particlebombardment (Cohen et al., supra, 1998). All transformations werecarried out under kanamycin selection (150 μg/ml) in which resistancewas conferred by the gene encoded by the segment in FIG. 2 labeled“Selection Marker.” (Chlamydomonas Stock Center, Duke University).

PCR was used to identify transformed strains. For PCR analysis, 10⁶algae cells (from agar plate or liquid culture) were suspended in 10 mMEDTA and heated to 95° C. for 10 minutes, then cooled to near 23° C. APCR cocktail consisting of reaction buffer, MgCl2, dNTPs, PCR primerpair(s) (Table 2 and shown graphically in FIG. 3A), DNA polymerase, andwater was prepared. Algae lysate in EDTA was added to provide templatefor reaction. Magnesium concentration is varied to compensate for amountand concentration of algae lysate in EDTA added. Annealing temperaturegradients were employed to determine optimal annealing temperature forspecific primer pairs.

To identify strains that contain the endo-β-glucanase gene, a primerpair was used in which one primer anneals to a site within the psbA5′UTR (SEQ ID NO. 1) and the other primer anneals within theendo-β-glucanase coding segment (SEQ ID NO. 3). Desired clones are thosethat yield a PCR product of expected size. To determine the degree towhich the endogenous gene locus is displaced (heteroplasmic vs.homoplasmic), a PCR reaction consisting of two sets of primer pairs wereemployed (in the same reaction). The first pair of primers amplifies theendogenous locus targeted by the expression vector and consists of aprimer that anneals within the psbA 5′UTR (SEQ ID NO. 8) and one thatanneals within the psbA coding region (SEQ ID NO. 9). The second pair ofprimers (SEQ ID NOs. 6 and 7) amplifies a constant, or control regionthat is not targeted by the expression vector, so should produce aproduct of expected size in all cases. This reaction confirms that theabsence of a PCR product from the endogenous locus did not result fromcellular and/or other contaminants that inhibited the PCR reaction.Concentrations of the primer pairs are varied so that both reactionswork in the same tube; however, the pair for the endogenous locus is 5×the concentration of the constant pair. The number of cycles usedwas >30 to increase sensitivity. The most desired clones are those thatyield a product for the constant region but not for the endogenous genelocus. Desired clones are also those that give weak-intensity endogenouslocus products relative to the control reaction.

Results from this PCR on 96 clones were determined and the results areshown in FIG. 4. FIG. 4A shows PCR results using the transgene-specificprimer pair. As can be seen, multiple transformed clones are positivefor insertion of the exo-β-glucanase gene (e.g. numbers 1-14). FIG. 4Bshows the PCR results using the primer pairs to differentiatehomoplasmic from heteroplasmic clones. As can be seen, multipletransformed clones are either homoplasmic or heteroplasmic to a degreein favor of incorporation of the transgene (e.g. numbers 1-14).Unnumbered clones demonstrate the presence of wild-type psbA and, thus,were not selected for further analysis.

TABLE 2 PCR primers. SEQ ID NO. Use Sequence 1. psbA 5′ UTR forwardprimer GTGCTAGGTAACTAACGTTTGATTTTT 2. Exo-β-glucanase reverse primerAACCTTCCACGTTAGCTTGA 3. Endo-β-glucanase reverse primerGCATTAGTTGGACCACCTTG 4. β-glucosidase reverse primerATCACCTGAAGCAGGTTTGA 5. Endoxylanase reverse primerGCACTACCTGATGAAAAATAACC 6. Control forward primer CCGAACTGAGGTTGGGTTTA7. Control reverse primer GGGGGAGCGAATAGGATTAG 8. psbA 5′ UTR forwardprimer (wild-type) GGAAGGGGACGTAGGTACATAAA 9. psbA 3′ reverse primer(wild-type) TTAGAACGTGTTTTGTTCCCAAT 10. psbC 5′ UTR forward primerTGGTACAAGAGGATTTTTGTTGTT 11. psbD 5′ UTR forward primerAAATTTAACGTAACGATGAGTTG 12. atpA 5′ UTR forward primerCCCCTTACGGGCAAGTAAAC 13. 3HB forward primer (wild-type)CTCGCCTATCGGCTAACAAG 14. 3HB forward primer (wild-type)CACAAGAAGCAACCCCTTGA

To ensure that the presence of the endo-β-glucanase-encoding gene led toexpression of the endo-β-glucanase protein, a Western blot wasperformed. Approximately 1×10⁸ algae cells were collected from TAP agarmedium and suspended in 0.5 ml of lysis buffer (750 mM Tris, pH=8.0, 15%sucrose, 100 mM beta-mercaptoethanol). Cells were lysed by sonication(5×30 sec at 15% power). Lysate was mixed 1:1 with loading buffer (5%SDS, 5% beta-mercaptoethanol, 30% sucrose, bromophenol blue) andproteins were separated by SDS-PAGE, followed by transfer to PVDFmembrane. The membrane was blocked with TBST+5% dried, nonfat milk at23° C. for 30 min, incubated with anti-FLAG antibody (diluted 1:1,000 inTBST+5% dried, nonfat milk) at 4° C. for 10 hours, washed three timeswith TBST, incubated with horseradish-linked anti-mouse antibody(diluted 1:10,000 in TBST+5% dried, nonfat milk) at 23° C. for 1 hour,and washed three times with TBST. Proteins were visualized withchemiluminescent detection. Results from multiple clones (FIG. 4C) showthat expression of the endo-β-glucanase gene in C. reinhardtii cellsresulted in production of the protein.

Cultivation of C. reinhardtii transformants for expression ofendo-β-glucanase was carried out in liquid TAP medium at 23° C. underconstant illumination of 5,000 Lux on a rotary shaker set at 100 rpm,unless stated otherwise. Cultures were maintained at a density of 1×10⁷cells per ml for at least 48 hr prior to harvest.

To determine if the endo-β-glucanase produced by transformed alga cellswas functional, endo-β-glucanase activity was tested using a filterpaper assay (Xiao et al., Biotech. Bioengineer. 88, 832-37, 2004).Briefly, 500 ml of algae cell culture was harvested by centrifugation at4000×g at 4° C. for 15 min. The supernatant was decanted and the cellsresuspended in 10 ml of lysis buffer (100 mM Tris-HCl, pH=8.0, 300 mMNaCl, 2% Tween-20). Cells were lysed by sonication (10×30 sec at 35%power). Lysate was clarified by centrifugation at 14,000×g at 4° C. for1 hour. The supernatant was removed and incubated with anti-FLAGantibody-conjugated agarose resin at 4° C. for 10 hours. Resin wasseparated from the lysate by gravity filtration and washed 3× with washbuffer ((100 mM Tris-HCl, pH=8.0, 300 mM NaCl, 2% Tween-20).Endo-β-glucanase was eluted by incubation of the resin with elutionbuffer (TBS, 250 ug/ml FLAG peptide). Results from Western blot analysisof samples collect after each step (FIG. 4D) show that theendo-β-glucanase protein was isolated. A 20 μl aliquot of diluted enzymewas added into wells containing 40 μl of 50 mM NaAc buffer and a filterpaper disk. After 60 minutes incubation at 50° C., 120 μl of DNS wasadded to each reaction and incubated at 95° C. for 5 minutes. Finally, a36 μl aliquot of each sample was transferred to the wells of aflat-bottom plate containing 160 μl water. The absorbance at 540 nm wasmeasured. The results for two transformed strains indicated that theisolated enzyme was functional (absorbance of 0.33 and 0.28).

Example 2 Production of Exo-β-glucanase in C. reinhardtii

In this example a nucleic acid encoding exo-β-glucanase from T. viridewas introduced into C. reinhardtii. Transforming DNA (SEQ ID NO. 19,Table 4) is shown graphically in FIG. 2A. In this instance the segmentlabeled “Transgene” is the exo-β-glucanase encoding gene (SEQ ID NO. 15,Table 3), the segment labeled “psbA 5′UTR” is the 5′ UTR and promotersequence for the psbA gene from C. reinhardtii, the segment labeled“psbA 3′UTR” contains the 3′ UTR for the psbA gene from C. reinhardtii,and the segment labeled “Selection Marker” is the kanamycin resistanceencoding gene from bacteria, which is regulated by the 5′ UTR andpromoter sequence for the atpA gene from C. reinhardtii and the 3′ UTRsequence for the rbcL gene from C. reinhardtii. The transgene cassetteis targeted to the psbA loci of C. reinhardtii via the segments labeled“5′Homology” and “3′ Homology,” which are identical to sequences of DNAflanking the psbA locus on the 5′ and 3′ sides, respectively. All DNAmanipulations carried out in the construction of this transforming DNAwere essentially as described by Sambrook et al., Molecular Cloning: ALaboratory Manual (Cold Spring Harbor Laboratory Press 1989) and Cohenet al., Meth. Enzymol. 297, 192-208, 1998.

For these experiments, all transformations were carried out on C.reinhardtii strain 137c (mt+). Cells were grown to late log phase(approximately 7 days) in the presence of 0.5 mM 5-fluorodeoxyuridine inTAP medium (Gorman and Levine, Proc. Natl. Acad. Sci., USA 54:1665-1669,1965, which is incorporated herein by reference) at 23° C. underconstant illumination of 450 Lux on a rotary shaker set at 100 rpm.Fifty ml of cells were harvested by centrifugation at 4,000×g at 23° C.for 5 min. The supernatant was decanted and cells resuspended in 4 mlTAP medium for subsequent chloroplast transformation by particlebombardment (Cohen et al., supra, 1998). All transformations werecarried out under kanamycin selection (150 μg/ml), in which resistancewas conferred by the gene encoded by the segment in FIG. 2 labeled“Selection Marker.” (Chlamydomonas Stock Center, Duke University).

PCR was used to identify transformed strains. For PCR analysis, 10⁶algae cells (from agar plate or liquid culture) were suspended in 10 mMEDTA and heated to 95° C. for 10 minutes, then cooled to near 23° C. APCR cocktail consisting of reaction buffer, MgCl2, dNTPs, PCR primerpair(s) (Table 2 and shown graphically in FIG. 3A), DNA polymerase, andwater was prepared. Algae lysate in EDTA was added to provide templatefor reaction. Magnesium concentration is varied to compensate for amountand concentration of algae lysate in EDTA added. Annealing temperaturegradients were employed to determine optimal annealing temperature forspecific primer pairs.

To identify strains that contain the exo-β-glucanase gene, a primer pairwas used in which one primer anneals to a site within the psbA 5′UTR(SEQ ID NO. 1) and the other primer anneals within the exo-β-glucanasecoding segment (SEQ ID NO. 2). Desired clones are those that yield a PCRproduct of expected size. To determine the degree to which theendogenous gene locus is displaced (heteroplasmic vs. homoplasmic), aPCR reaction consisting of two sets of primer pairs were employed (inthe same reaction). The first pair of primers amplifies the endogenouslocus targeted by the expression vector and consists of a primer thatanneals within the psbA 5′UTR (SEQ ID NO. 8) and one that anneals withinthe psbA coding region (SEQ ID NO. 9). The second pair of primers (SEQID NOs. 6 and 7) amplifies a constant, or control region that is nottargeted by the expression vector, so should produce a product ofexpected size in all cases. This reaction confirms that the absence of aPCR product from the endogenous locus did not result from cellularand/or other contaminants that inhibited the PCR reaction.Concentrations of the primer pairs are varied so that both reactionswork in the same tube; however, the pair for the endogenous locus is 5×the concentration of the constant pair. The number of cycles usedwas >30 to increase sensitivity. The most desired clones are those thatyield a product for the constant region but not for the endogenous genelocus. Desired clones are also those that give weak-intensity endogenouslocus products relative to the control reaction.

Results from this PCR on 96 clones were determined and the results areshown in FIG. 5. FIG. 5A shows PCR results using the transgene-specificprimer pair. As can be seen, multiple transformed clones are positivefor insertion of the endo-β-glucanase gene (e.g. numbers 1-14). FIG. 4Bshows the PCR results using the primer pairs to differentiatehomoplasmic from heteroplasmic clones. As can be seen, multipletransformed clones are either homoplasmic or heteroplasmic to a degreein favor of incorporation of the transgene (e.g. numbers 1-14).Unnumbered clones demonstrate the presence of wild-type psbA and, thus,were not selected for further analysis.

To ensure that the presence of the exo-β-glucanase-encoding gene led toexpression of the exo-β-glucanase protein, a Western blot was performed.Approximately 1×10⁸ algae cells were collected from TAP agar medium andsuspended in 0.5 ml of lysis buffer (750 mM Tris, pH=8.0, 15% sucrose,100 mM beta-mercaptoethanol). Cells were lysed by sonication (5×30 secat 15% power). Lysate was mixed 1:1 with loading buffer (5% SDS, 5%beta-mercaptoethanol, 30% sucrose, bromophenol blue) and proteins wereseparated by SDS-PAGE, followed by transfer to PVDF membrane. Themembrane was blocked with TBST+5% dried, nonfat milk at 23° C. for 30min, incubated with anti-FLAG antibody (diluted 1:1,000 in TBST+5%dried, nonfat milk) at 4° C. for 10 hours, washed three times with TBST,incubated with horseradish-linked anti-mouse antibody (diluted 1:10,000in TBST+5% dried, nonfat milk) at 23° C. for 1 hour, and washed threetimes with TBST. Proteins were visualized with chemiluminescentdetection. Results from multiple clones (FIG. 5C) show that expressionof the exo-β-glucanase gene in C. reinhardtii cells resulted inproduction of the protein.

Cultivation of C. reinhardtii transformants for expression ofendo-β-glucanase was carried out in liquid TAP medium at 23° C. underconstant illumination of 5,000 Lux on a rotary shaker set at 100 rpm,unless stated otherwise. Cultures were maintained at a density of 1×10⁷cells per ml for at least 48 hr prior to harvest.

To determine if the exo-β-glucanase produced by transformed alga cellswas functional, exo-β-glucanase activity was tested using a filter paperassay (Xiao et al., Biotech. Bioengineer. 88, 832-37, 2004). Briefly,500 ml of algae cell culture was harvested by centrifugation at 4000×gat 4° C. for 15 min. The supernatant was decanted and the cellsresuspended in 10 ml of lysis buffer (100 mM Tris-HCl, pH=8.0, 300 mMNaCl, 2% Tween-20). Cells were lysed by sonication (10×30 sec at 35%power). Lysate was clarified by centrifugation at 14,000×g at 4° C. for1 hour. The supernatant was removed and incubated with anti-FLAGantibody-conjugated agarose resin at 4° C. for 10 hours. Resin wasseparated from the lysate by gravity filtration and washed 3× with washbuffer (100 mM Tris-HCl, pH=8.0, 300 mM NaCl, 2% Tween-20).Exo-β-glucanase was eluted by incubation of the resin with elutionbuffer (TBS, 250 ug/ml FLAG peptide). Results from Western blot analysisof samples collect after each step (FIG. 5D) show that theexo-β-glucanase protein was isolated. A 20 μl aliquot of diluted enzymewas added into wells containing 40 μl of 50 mM NaAc buffer and a filterpaper disk. After 60 minutes incubation at 50° C., 120 μl of DNS wasadded to each reaction and incubated at 95° C. for 5 minutes. Finally, a36 μl aliquot of each sample was transferred to the wells of aflat-bottom plate containing 160 μl water. The absorbance at 540 nm wasmeasured. The results for two transformed strains indicated that theisolated enzyme was functional (absorbance of 0.20 and 0.45).

Example 3 Production of α-Glucosidase in C. reinhardti

In this example a nucleic acid encoding β-glucosidase from T. reesei wasintroduced into C. reinhardtii. Transforming DNA (SEQ ID NO. 21, Table4) is shown graphically in FIG. 2A. The amino acid sequence encoded bythis gene is shown in Table 3. In this instance the segment labeled“Transgene” is the β-glucosidase encoding gene (SEQ ID NO. 17, Table 3),the segment labeled “psbA 5′UTR” is the 5′ UTR and promoter sequence forthe psbA gene from C. reinhardtii, the segment labeled “psbA 3′UTR”contains the 3′ UTR for the psbA gene from C. reinhardtii, and thesegment labeled “Selection Marker” is the kanamycin resistance encodinggene from bacteria, which is regulated by the 5′ UTR and promotersequence for the atpA gene from C. reinhardtii and the 3′ UTR sequencefor the rbcL gene from C. reinhardtii. The transgene cassette istargeted to the psbA loci of C. reinhardtii via the segments labeled“5′Homology” and “3′ Homology,” which are identical to sequences of DNAflanking the psbA locus on the 5′ and 3′ sides, respectively. All DNAmanipulations carried out in the construction of this transforming DNAwere essentially as described by Sambrook et al., Molecular Cloning: ALaboratory Manual (Cold Spring Harbor Laboratory Press 1989) and Cohenet al., Meth. Enzymol. 297, 192-208, 1998.

For these experiments, all transformations were carried out on C.reinhardtii strain 137c (mt+). Cells were grown to late log phase(approximately 7 days) in the presence of 0.5 mM 5-fluorodeoxyuridine inTAP medium (Gorman and Levine, Proc. Natl. Acad. Sci., USA 54:1665-1669,1965, which is incorporated herein by reference) at 23° C. underconstant illumination of 450 Lux on a rotary shaker set at 100 rpm.Fifty ml of cells were harvested by centrifugation at 4,000×g at 23° C.for 5 min. The supernatant was decanted and cells resuspended in 4 mlTAP medium for subsequent chloroplast transformation by particlebombardment (Cohen et al., supra, 1998). All transformations werecarried out under kanamycin selection (150 μg/ml), in which resistancewas conferred by the gene encoded by the segment in FIG. 2 labeled“Selection Marker.” (Chlamydomonas Stock Center, Duke University).

PCR was used to identify transformed strains. For PCR analysis, 10⁶algae cells (from agar plate or liquid culture) were suspended in 10 mMEDTA and heated to 95° C. for 10 minutes, then cooled to near 23° C. APCR cocktail consisting of reaction buffer, MgCl2, dNTPs, PCR primerpair(s) (Table 2 and shown graphically in FIG. 3A), DNA polymerase, andwater was prepared. Algae lysate in EDTA was added to provide templatefor reaction. Magnesium concentration is varied to compensate for amountand concentration of algae lysate in EDTA added. Annealing temperaturegradients were employed to determine optimal annealing temperature forspecific primer pairs.

To identify strains that contain the β-glucosidase gene, a primer pairwas used in which one primer anneals to a site within the psbA 5′UTR(SEQ ID NO. 1) and the other primer anneals within the β-glucosidasecoding segment (SEQ ID NO. 4). Desired clones are those that yield a PCRproduct of expected size. To determine the degree to which theendogenous gene locus is displaced (heteroplasmic vs. homoplasmic), aPCR reaction consisting of two sets of primer pairs were employed (inthe same reaction). The first pair of primers amplifies the endogenouslocus targeted by the expression vector and consists of a primer thatanneals within the psbA 5′UTR (SEQ ID NO. 8) and one that anneals withinthe psbA coding region (SEQ ID NO. 9). The second pair of primers (SEQID NOs. 6 and 7) amplifies a constant, or control region that is nottargeted by the expression vector, so should produce a product ofexpected size in all cases. This reaction confirms that the absence of aPCR product from the endogenous locus did not result from cellularand/or other contaminants that inhibited the PCR reaction.Concentrations of the primer pairs are varied so that both reactionswork in the same tube; however, the pair for the endogenous locus is 5×the concentration of the constant pair. The number of cycles usedwas >30 to increase sensitivity. The most desired clones are those thatyield a product for the constant region but not for the endogenous genelocus. Desired clones are also those that give weak-intensity endogenouslocus products relative to the control reaction.

Results from this PCR on 96 clones were determined and the results areshown in FIG. 6. FIG. 6A shows PCR results using the transgene-specificprimer pair. As can be seen, multiple transformed clones are positivefor insertion of the endo-β-glucanase gene (e.g. numbers 1-9). FIG. 6Bshows the PCR results using the primer pairs to differentiatehomoplasmic from heteroplasmic clones. As can be seen, multipletransformed clones are either homoplasmic or heteroplasmic to a degreein favor of incorporation of the transgene (e.g. numbers 1-9).Unnumbered clones demonstrate the presence of wild-type psbA and, thus,were not selected for further analysis.

To ensure that the presence of the β-glucosidase-encoding gene led toexpression of the β-glucosidase protein, a Western blot was performed.Approximately 1×10⁸ algae cells were collected from TAP agar medium andsuspended in 0.5 ml of lysis buffer (750 mM Tris, pH=8.0, 15% sucrose,100 mM beta-mercaptoethanol). Cells were lysed by sonication (5×30 secat 15% power). Lysate was mixed 1:1 with loading buffer (5% SDS, 5%beta-mercaptoethanol, 30% sucrose, bromophenol blue) and proteins wereseparated by SDS-PAGE, followed by transfer to PVDF membrane. Themembrane was blocked with TBST+5% dried, nonfat milk at 23° C. for 30min, incubated with anti-FLAG antibody (diluted 1:1,000 in TBST+5%dried, nonfat milk) at 4° C. for 10 hours, washed three times with TBST,incubated with horseradish-linked anti-mouse antibody (diluted 1:10,000in TBST+5% dried, nonfat milk) at 23° C. for 1 hour, and washed threetimes with TBST. Proteins were visualized with chemiluminescentdetection. Results from multiple clones (FIG. 6C) show that expressionof the β-glucosidase gene in C. reinhardtii cells resulted in productionof the protein.

To determine if the β-glucosidase produced by transformed alga cells wasfunctional, β-glucosidase activity was tested using an enzyme functionassay. Briefly, 500 ml of algae cell culture was harvested bycentrifugation at 4000×g at 4° C. for 15 min. The supernatant wasdecanted and the cells resuspended in 10 ml of lysis buffer (100 mMTris-HCl, pH=8.0, 300 mM NaCl, 2% Tween-20). Cells were lysed bysonication (10×30 sec at 35% power). Lysate was clarified bycentrifugation at 14,000×g at 4° C. for 1 hour. The supernatant wasremoved and incubated with anti-FLAG antibody-conjugated agarose resinat 4° C. for 10 hours. Resin was separated from the lysate by gravityfiltration and washed 3× with wash buffer ((100 mM Tris-HCl, pH=8.0, 300mM NaCl, 2% Tween-20). β-glucosidase was eluted by incubation of theresin with elution buffer (TBS, 250 ug/ml FLAG peptide). Western blotanalysis of samples collect after each step (FIG. 6D) show that theβ-glucosidase protein was isolated. For each sample tested, 50 μl ofp-Nitrophenyl-/3-D-glucoside (substrate), 90 μl of 0.1 M sodium acetatebuffer (pH 4.8), and 10 μl enzyme was added to a microplate well. Thereaction was incubated at 50° C. for one hour and then the reaction wasstopped with a glycine buffer. The absorbance of the liberatedp-nitrophenol was measured at 430 nm. The results for two transformedstrains indicated that the isolated enzyme was functional (absorbance of0.157 and 0.284).

Example 4 Production of Endoxylanase in C. reinhardtii

In this example a nucleic acid encoding endoxylanase from T. reesei wasintroduced into C. reinhardtii. Transforming DNA (SEQ ID NO. 22, Table4) is shown graphically in FIG. 2A. The amino acid sequence encoded bythis gene is shown in Table 3. In this instance the segment labeled“Transgene” is the endoxylanase encoding gene (SEQ ID NO. 18, Table 3),the segment labeled “psbA 5′UTR” is the 5′ UTR and promoter sequence forthe psbA gene from C. reinhardtii, the segment labeled “psbA 3′UTR”contains the 3′ UTR for the psbA gene from C. reinhardtii, and thesegment labeled “Selection Marker” is the kanamycin resistance encodinggene from bacteria, which is regulated by the 5′ UTR and promotersequence for the atpA gene from C. reinhardtii and the 3′ UTR sequencefor the rbcL gene from C. reinhardtii. The transgene cassette istargeted to the psbA loci of C. reinhardtii via the segments labeled“5′Homology” and “3′ Homology,” which are identical to sequences of DNAflanking the psbA locus on the 5′ and 3′ sides, respectively. All DNAmanipulations carried out in the construction of this transforming DNAwere essentially as described by Sambrook et al., Molecular Cloning: ALaboratory Manual (Cold Spring Harbor Laboratory Press 1989) and Cohenet al., Meth. Enzymol. 297, 192-208, 1998.

For these experiments, all transformations were carried out on C.reinhardtii strain 137c (mt+). Cells were grown to late log phase(approximately 7 days) in the presence of 0.5 mM 5-fluorodeoxyuridine inTAP medium (Gorman and Levine, Proc. Natl. Acad. Sci., USA 54:1665-1669,1965, which is incorporated herein by reference) at 23° C. underconstant illumination of 450 Lux on a rotary shaker set at 100 rpm.Fifty ml of cells were harvested by centrifugation at 4,000×g at 23° C.for 5 min. The supernatant was decanted and cells resuspended in 4 mlTAP medium for subsequent chloroplast transformation by particlebombardment (Cohen et al., supra, 1998). All transformations werecarried out under kanamycin selection (150 μg/ml), in which resistancewas conferred by the gene encoded by the segment in FIG. 2 labeled“Selection Marker.” (Chlamydomonas Stock Center, Duke University).

PCR was used to identify transformed strains. For PCR analysis, 106algae cells (from agar plate or liquid culture) were suspended in 10 mMEDTA and heated to 95° C. for 10 minutes, then cooled to near 23° C. APCR cocktail consisting of reaction buffer, MgCl2, dNTPs, PCR primerpair(s) (Table 2 and shown graphically in FIG. 3A), DNA polymerase, andwater was prepared. Algae lysate in EDTA was added to provide templatefor reaction. Magnesium concentration is varied to compensate for amountand concentration of algae lysate in EDTA added. Annealing temperaturegradients were employed to determine optimal annealing temperature forspecific primer pairs.

To identify strains that contain the endoxylanase gene, a primer pairwas used in which one primer anneals to a site within the psbA 5′UTR(SEQ ID NO. 1) and the other primer anneals within the endoxylanasecoding segment (SEQ ID NO. 5). Desired clones are those that yield a PCRproduct of expected size. To determine the degree to which theendogenous gene locus is displaced (heteroplasmic vs. homoplasmic), aPCR reaction consisting of two sets of primer pairs were employed (inthe same reaction). The first pair of primers amplifies the endogenouslocus targeted by the expression vector and consists of a primer thatanneals within the psbA 5′UTR (SEQ ID NO. 8) and one that anneals withinthe psbA coding region (SEQ ID NO. 9). The second pair of primers (SEQID NOs. 6 and 7) amplifies a constant, or control region that is nottargeted by the expression vector, so should produce a product ofexpected size in all cases. This reaction confirms that the absence of aPCR product from the endogenous locus did not result from cellularand/or other contaminants that inhibited the PCR reaction.Concentrations of the primer pairs are varied so that both reactionswork in the same tube; however, the pair for the endogenous locus is 5×the concentration of the constant pair. The number of cycles usedwas >30 to increase sensitivity. The most desired clones are those thatyield a product for the constant region but not for the endogenous genelocus. Desired clones are also those that give weak-intensity endogenouslocus products relative to the control reaction.

Results from this PCR on 96 clones were determined and the results areshown in FIG. 7. FIG. 7A shows PCR results using the transgene-specificprimer pair. As can be seen, multiple transformed clones are positivefor insertion of the endo-β-glucanase gene (e.g. numbers 1-9). FIG. 7Bshows the PCR results using the primer pairs to differentiatehomoplasmic from heteroplasmic clones. As can be seen, multipletransformed clones are either homoplasmic or heteroplasmic to a degreein favor of incorporation of the transgene (e.g. numbers 1-9).Unnumbered clones demonstrate the presence of wild-type psbA and, thus,were not selected for further analysis.

To ensure that the presence of the endoxylanase-encoding gene led toexpression of the endoxylanase protein, a Western blot was performed.Approximately 1×10⁸ algae cells were collected from TAP agar medium andsuspended in 0.5 ml of lysis buffer (750 mM Tris, pH=8.0, 15% sucrose,100 mM beta-mercaptoethanol). Cells were lysed by sonication (5×30 secat 15% power). Lysate was mixed 1:1 with loading buffer (5% SDS, 5%beta-mercaptoethanol, 30% sucrose, bromophenol blue) and proteins wereseparated by SDS-PAGE, followed by transfer to PVDF membrane. Themembrane was blocked with TBST+5% dried, nonfat milk at 23° C. for 30min, incubated with anti-FLAG antibody (diluted 1:1,000 in TBST+5%dried, nonfat milk) at 4° C. for 10 hours, washed three times with TBST,incubated with horseradish-linked anti-mouse antibody (diluted 1:10,000in TBST+5% dried, nonfat milk) at 23° C. for 1 hour, and washed threetimes with TBST. Proteins were visualized with chemiluminescentdetection. Results from multiple clones (FIG. 7C) show that expressionof the endoxylanase gene in C. reinhardtii cells resulted in productionof the protein.

To determine if the endoxylanase produced by transformed alga cells wasfunctional, endoxylanase activity was tested using an enzyme functionassay. Briefly, 500 ml of algae cell culture was harvested bycentrifugation at 4000×g at 4° C. for 15 min. The supernatant wasdecanted and the cells resuspended in 10 ml of lysis buffer (100 mMTris-HCl, pH=8.0, 300 mM NaCl, 2% Tween-20). Cells were lysed bysonication (10×30 sec at 35% power). Lysate was clarified bycentrifugation at 14,000×g at 4° C. for 1 hour. The supernatant wasremoved and incubated with anti-FLAG antibody-conjugated agarose resinat 4° C. for 10 hours. Resin was separated from the lysate by gravityfiltration and washed 3× with wash buffer ((100 mM Tris-HCl, pH=8.0, 300mM NaCl, 2% Tween-20). Endoxylanase was eluted by incubation of theresin with elution buffer (TBS, 250 ug/ml FLAG peptide). Results fromWestern blot analysis of samples collect after each step (FIG. 7D) showthat the Endoxylanase protein was isolated. To test for enzyme function,0.5 ml aliquots of diluted enzyme preparation were added to glass testtubes and equilibrated at 40° C. for 5 minutes. A Xylazyme AX testtablet (Megazyme) was added to initiate the reaction. After 30 minutes,the reaction was terminated by adding 10 ml Trizma base solution withvigorous stirring. The tubes were incubated at room temperature for 5minutes and the reaction was stirred again. The reaction was thenfiltered through a Whatman No. 1 (9 cm) filter circle. The filtrate wasthen clarified by microcentrifugation. The absorbance of the filtratewas measured at 590 nm. The results indicate that, for crude enzymeextracts from two different clones, endoxylanase activity was present(absorbance of 0.974 and 0.488).

Example 5 Determination of Level of Protein Expression in a C.reinhardtii Strain Producing Exogneous Endo-β-Glucanase

Western blot analysis of proteins was done as follows. Approximately1×10⁸ algae cells were collected from liquid cultures growing in TAPmedium at 23° C. under constant illumination of 5,000 Lux on a rotaryshaker set at 100 rpm. Cells were suspended in 0.5 ml of lysis buffer(750 mM Tris, pH=8.0, 15% sucrose, 100 mM beta-mercaptoethanol) andlysed by sonication (5×30 sec at 15% power). Lysates were centrifuged at14,000 RPM for 15 minutes at 4° C. and the supernatant was collected.Total soluble protein concentrations were determined using BioRad'sprotein assay reagent. The sample concentrations were then normalized toone another. The FLAG control protein was a FLAG tagged bacterialalkaline phosphatase protein standard (Sigma-Aldrich, St. Louis, Mo.).Lysate was mixed 1:1 with loading buffer (5% SDS, 5%beta-mercaptoethanol, 30% sucrose, bromophenol blue) and proteins wereseparated by SDS-PAGE, followed by transfer to PVDF membrane. Themembrane was blocked with TBST+5% dried, nonfat milk at 23° C. for 30min, incubated with anti-FLAG antibody (diluted 1:1,000 in TBST+5%dried, nonfat milk) at 4° C. for 10 hours, washed three times with TBST,incubated with horseradish-linked anti-mouse antibody (diluted 1:10,000in TBST+5% dried, nonfat milk) at 23° C. for 1 hour, and washed threetimes with TBST. Proteins were visualized with chemiluminescentdetection.

To ascertain the level of cellulase accumulating in the transformantsunder different growth conditions, we carried out the titration shown inFIG. 8. Five, ten and twenty μg of total protein from a transformantexpressing endo-B-glucanase (BD5-26) were separated along with 10, 50,100 and 200 ug of a control protein. Both proteins contain the FLAGepitope tag on their carboxy terminus, thus a direct comparison can bemade between the two proteins to determine expression levels. Acomparison of the signal intensity between the 5 ug samples form either24 or 48 hours growth, show a signal greater than the 50 ng controlpeptide and close in intensity to the 100 ng sample. A 1% total proteinexpression level would equal 1/100 or 50 ng of a 5 ug sample. Theintensity here shows a signal equal to a level of twice that, or 100 ngin the 5 ug sample which is equal to 2% of total protein.

TABLE 3 Amino Acid Sequences of Cellulolytic Enzymes. SEQ ID NO.Sequence Source 15MVPYRKLAVISAFLATARAQSACTLQSETHPPLTWQKCSSGGTCTQQTGSVVIDANWRWTHATNSSTNCYDGNTWSSTExo-β-LCPDNETCAKNCCLDGAAYASTYGVTTSGNSLSIGFVTQSAQKNVGARLYLMASDTTYQEFTLLGNEFSFDVDVSQLPCglucanaseGLNGALYFVSMDADGGVSKYPTNTAGAKYGTGYCDSQCPRDLKFINGQANVEGWEPSSNNANTGIGGHGSCCSEMDIWfrom T. virideEANSISEALTPHPCTTVGQEICEGDGCGGTYSDNRYGGTCDPDGCDWDPYRLGNTSFYGPGSSFTLDTTKKLTVVTQFETSGAINRYYVQNGVTFQQPNAELGSYSGNGLNDDYCTAEEAEFGGSSFSDKGGLTQFKKATSGGMVLVMSLWDDYYANMLWLDSTYPTNETSSTPGAVRGSCSTSSGVPAQVESQSPNAKVTFSNIKFGPIGSTGDPSGGNPPGGNPPGTTTTRRPATTTGSSPGPTQSHYGQCGGIGYSGPTVCASGTTCQVLNPYYSQCLGTGENLYFQGSGGGGSDYKDDDDKGTG 16MVPNKSVAPLLLAASILYGGAVAQQTVWGQCGGIGWSGPTNCAPGSACSTLNPYYAQCIPGATTITTSTRPPSGPTTTTRAEndo-β-TSTSSSTPPTSSGVRFAGVNIAGFDFGCTTDGTCVTSKVYPPLKNFTGSNNYPDGIGQMQHFVNEDGMTIFRLPVGWQYLVglucanaseNNNLGGNLDSTSISKYDQLVQGCLSLGAYCIVDIHNYARWNGGIIGQGGPTNAQFTSLWSQLASKYASQSRVWFGIMNEPfrom T. reeseiHDVNINTWAATVQEVVTAIRNAGATSQFISLPGNDWQSAGAFISDGSAAALSQVTNPDGSTTNLIFDVHKYLDSDNSGTHAECTTNNIDGAFSPLATWLRQNNRQAILTETGGGNVQSCIQDMCQQIQYLNQNSDVYLGYVGWGAGSFDSTYVLTETPTSSGNSWTDTSLVSSCLARKGTGENLYFQGSGGGGSDYKDDDDKGTG 17MVPLPKDFQWGFATAAYQIEGAVDQDGRGPSIWDTFCAQPGKIADGSSGVTACDSYNRTAEDIALLKSLGAKSYRFSISWSβ-glucosidaseRIIPEGGRGDAVNQAGIDHYVKFVDDLLDAGITPFITLFHWDLPEGLHQRYGGLLNRTEFPLDFENYARVMFRALPKVRNWIfrom T. reeseiTFNEPLCSAIPGYGSGTFAPGRQSTSEPWTVGHNILVAHGRAVKAYRDDFKPASGDGQIGIVLNGDFTYPWDAADPADKEAAERRLEFFTAWFADPIYLGDYPASMRKQLGDRLPTFTPEERALVHGSNDFYGMNHYTSNYIRHRSSPASADDTVGNVDVLFTNKQGNCIGPETQSPWLRPCAAGFRDFLVWISKRYGYPPIYVTENGTSIKGESDLPKEKILEDDFRVKYYNEYIRAMVTAVELDGVNVKGYFAWSLMDNFEWADGYVTRFGVTYVDYENGQKRFPKKSAKSLKPLFDELIAAAGTGENLYFQGSGGGGSDYKDDDDKGTG 18MVPVSFTSLLAASPPSRASCRPAAEVESVAVEKRQTIQPGTGYNNGYFYSYWNDGHGGVTYTNGPGGQFSVNWSNSGNFVGEndo-GKGWQPGTKNKVINFSGSYNPNGNSYLSVYGWSRNPLIEYYIVENFGTYNPSTGATKLGEVTSDGSVYDIYRTQRVNQPSIIGxylanaseTATFYQYWSVRRNHRSSGSVNTANHFNAWAQQGLTLGTMDYQIVAVEGYFSSGSASITVSGTGENLYFQGSGGGGSDYKDDfrom T. reesei DDKGTG

Example 6 Construction of a C. reinhardtii Strain Transformed withMultiple Biodegradative Enzyme-Encoding Genes

In this example a strain containing multiple biomass degrading (BD)enzyme-encoding genes using two separate constructs is described. One ofskill in the art will realize that such an approach is provided merelyby way of example. Transformation of a strain with a single constructcontaining all the genes of interest is performed generally as describedin prior examples. An example of constructs which could be used totransform such a strain is shown in FIG. 9. As can be seen in thefigure, two polynucleotides are constructed for the delivery of multiplegenes into a host alga cell. The upper construct contains threeenzyme-coding sequences (FIG. 9 BD-5, BD-1, and BD-9). The lowerconstruct contains three enzyme-coding sequences (FIG. 9 BD-2, BD-4, andBD-11). The numbers used in this figure are meant only to indicate thatdifferent enzymes are encoded by each gene. In some instances, the genesencode different enzymes in one or more biomass degrading pathways. Inother instances, one or more of the genes encode the same enzyme, butone may be a mutated form or some may be from multiple organisms. Bothconstructs contain terminal regions which have homology to the C.reinhardtii genome to facilitate integration into the chloroplastgenome. Proper transformation, integration, protein production andprotein function is analyzed as described above.

Each construct contains a selectable marker (FIG. 9 Marker I and MarkerII). The C. reinhardtii cells are transformed as described above.Introduction of the two constructs can be by co-transformation with bothconstructs. In such instances, potential transformants are selected bygrowth on TAP medium supplemented with substances which will select forthe presence of both markers (e.g., streptomycin and kanamycinresistance).

The genes of both constructs may be placed under control of a singletranscriptional control, in essence introducing a synthetic operon(“chloroperon”) into the chloroplasts of the alga cells. Such anapproach allows for an entire pathway to be engineered into achloroplast. Alternately, the separate constructs may be placed undercontrol of different transcriptional regulators. Additionally, each geneso introduced may be placed under control of different transcriptionalregulators.

Example 7 Construction of a C. reinhardtii Strain Transformed with aConstruct that does not Disrupt Photosynthetic Capability

In this example a nucleic acid encoding endo-β-glucanase from T. reeseiwas introduced into C. reinhardtii. Transforming DNA (SEQ ID NO. 30,Table 4) is shown graphically in FIG. 2B. In this instance the segmentlabeled “Transgene” is the endo-β-glucanase encoding gene (SEQ ID NO.16, Table 3), the segment labeled 5′ UTR is the 5′ UTR and promotersequence for the psbD gene from C. reinhardtii, the segment labeled 3′UTR contains the 3′ UTR for the psbA gene from C. reinhardtii, and thesegment labeled “Selection Marker” is the kanamycin resistance encodinggene from bacteria, which is regulated by the 5′ UTR and promotersequence for the atpA gene from C. reinhardtii and the 3′ UTR sequencefor the rbcL gene from C. reinhardtii. The transgene cassette istargeted to the 3HB locus of C. reinhardtii via the segments labeled“5′Homology” and “3′ Homology,” which are identical to sequences of DNAflanking the 3HB locus on the 5′ and 3′ sides, respectively. All DNAmanipulations carried out in the construction of this transforming DNAwere essentially as described by Sambrook et al., Molecular Cloning: ALaboratory Manual (Cold Spring Harbor Laboratory Press 1989) and Cohenet al., Meth. Enzymol. 297, 192-208, 1998.

For these experiments, all transformations were carried out on C.reinhardtii strain 137c (mt+). Cells were grown to late log phase(approximately 7 days) in the presence of 0.5 mM 5-fluorodeoxyuridine inTAP medium (Gorman and Levine, Proc. Natl. Acad. Sci., USA 54:1665-1669,1965, which is incorporated herein by reference) at 23° C. underconstant illumination of 450 Lux on a rotary shaker set at 100 rpm.Fifty ml of cells were harvested by centrifugation at 4,000×g at 23° C.for 5 min. The supernatant was decanted and cells resuspended in 4 mlTAP medium for subsequent chloroplast transformation by particlebombardment (Cohen et al., supra, 1998). All transformations werecarried out under kanamycin selection (100 μg/ml), in which resistancewas conferred by the gene encoded by the segment in FIG. 2B labeled“Selection Marker.” (Chlamydomonas Stock Center, Duke University).

PCR was used to identify transformed strains. For PCR analysis, 10⁶algae cells (from agar plate or liquid culture) were suspended in 10 mMEDTA and heated to 95° C. for 10 minutes, then cooled to near 23° C. APCR cocktail consisting of reaction buffer, MgCl2, dNTPs, PCR primerpair(s) (Table 2 and shown graphically in FIG. 3B), DNA polymerase, andwater was prepared. Algae lysate in EDTA was added to provide templatefor reaction. Magnesium concentration is varied to compensate for amountand concentration of algae lysate in EDTA added. Annealing temperaturegradients were employed to determine optimal annealing temperature forspecific primer pairs.

To identify strains that contain the endo-β-glucanase gene, a primerpair was used in which one primer anneals to a site within the psbD5′UTR (SEQ ID NO. 11) and the other primer anneals within theendo-β-glucanase coding segment (SEQ ID NO. 3). Desired clones are thosethat yield a PCR product of expected size. To determine the degree towhich the endogenous gene locus is displaced (heteroplasmic vs.homoplasmic), a PCR reaction consisting of two sets of primer pairs wereemployed (in the same reaction). The first pair of primers amplifies theendogenous locus targeted by the expression vector (SEQ ID NOs. 13 and14). The second pair of primers (SEQ ID NOs. 6 and 7) amplifies aconstant, or control region that is not targeted by the expressionvector, so should produce a product of expected size in all cases. Thisreaction confirms that the absence of a PCR product from the endogenouslocus did not result from cellular and/or other contaminants thatinhibited the PCR reaction. Concentrations of the primer pairs arevaried so that both reactions work in the same tube; however, the pairfor the endogenous locus is 5× the concentration of the constant pair.The number of cycles used was >30 to increase sensitivity. The mostdesired clones are those that yield a product for the constant regionbut not for the endogenous gene locus. Desired clones are also thosethat give weak-intensity endogenous locus products relative to thecontrol reaction.

Results from this PCR on 96 clones were determined and the results areshown in FIG. 14. FIG. 14A shows PCR results using thetransgene-specific primer pair. As can be seen, multiple transformedclones are positive for insertion of the endo-β-glucanase gene (e.g.numbers 1, 4, and 14). FIG. 14B shows the PCR results using the primerpairs to differentiate homoplasmic from heteroplasmic clones. As can beseen, multiple transformed clones are either homoplasmic orheteroplasmic to a degree in favor of incorporation of the transgene(e.g. numbers 1, 4, and 14). Unnumbered clones demonstrate the presenceof wild-type psbA and, thus, were not selected for further analysis.

To ensure that the presence of the endo-β-glucanase-encoding gene led toexpression of the endo-β-glucanase protein, a Western blot wasperformed. Approximately 1×10⁸ algae cells were collected from TAP agarmedium and suspended in 0.5 ml of lysis buffer (750 mM Tris, pH=8.0, 15%sucrose, 100 mM beta-mercaptoethanol). Cells were lysed by sonication(5×30 sec at 15% power). Lysate was mixed 1:1 with loading buffer (5%SDS, 5% beta-mercaptoethanol, 30% sucrose, bromophenol blue) andproteins were separated by SDS-PAGE, followed by transfer to PVDFmembrane. The membrane was blocked with TBST+5% dried, nonfat milk at23° C. for 30 min, incubated with anti-FLAG antibody (diluted 1:1,000 inTBST+5% dried, nonfat milk) at 4° C. for 10 hours, washed three timeswith TBST, incubated with horseradish-linked anti-mouse antibody(diluted 1:10,000 in TBST+5% dried, nonfat milk) at 23° C. for 1 hour,and washed three times with TBST. Proteins were visualized withchemiluminescent detection. Results from multiple clones (FIG. 14C) showthat expression of the endo-β-glucanase gene in C. reinhardtii cellsresulted in production of the protein.

Similar results were seen (FIG. 15) with a similar construct containingthe endoxylanase gene from T. reesei (SEQ ID NO. 31, Table 4). Theconstruct containing the endoxylanase gene is depicted in FIG. 2B. Inthis instance the segment labeled “Transgene” is the endoxylanaseencoding gene (SEQ ID NO. 18, Table 3), the segment labeled 5′ UTR isthe 5′ UTR and promoter sequence for the psbD gene from C. reinhardtii,the segment labeled 3′ UTR contains the 3′ UTR for the psbA gene from C.reinhardtii, and the segment labeled “Selection Marker” is the kanamycinresistance encoding gene from bacteria, which is regulated by the 5′ UTRand promoter sequence for the atpA gene from C. reinhardtii and the 3′UTR sequence for the rbcL gene from C. reinhardtii. The transgenecassette is targeted to the 3HB locus of C. reinhardtii via the segmentslabeled “5′Homology” and “3′ Homology,” which are identical to sequencesof DNA flanking the 3HB locus on the 5′ and 3′ sides, respectively. AllDNA manipulations carried out in the construction of this transformingDNA were essentially as described by Sambrook et al., Molecular Cloning:A Laboratory Manual (Cold Spring Harbor Laboratory Press 1989) and Cohenet al., Meth. Enzymol. 297, 192-208, 1998.

FIG. 15A shows PCR using the gene-specific primer pair. As can be seen,multiple transformed clones are positive for insertion of theendoxylanase gene. FIG. 15B shows the PCR results using the primer pairsto differentiate homoplasmic from heteroplasmic clones. As can be seen,multiple transformed clones are either homoplasmic or heteroplasmic to adegree in favor of incorporation of the transgene. Unnumbered clonesdemonstrate the presence of wild-type psbA and, thus, were not selectedfor further analysis. Western blot analysis demonstrating proteinexpression is demonstrated in FIG. 15C.

Similar results were seen (FIG. 16) with a similar construct containingthe exo-β-glucanase gene from T. viride (SEQ ID NO. 29, Table 4). Theconstruct containing the exo-β-glucanase gene is depicted in FIG. 2B. Inthis instance the segment labeled “Transgene” is the exo-β-glucanaseencoding gene (SEQ ID NO. 15, Table 3), the segment labeled 5′ UTR isthe 5′ UTR and promoter sequence for the psbD gene from C. reinhardtii,the segment labeled 3′ UTR contains the 3′ UTR for the psbA gene from C.reinhardtii, and the segment labeled “Selection Marker” is the kanamycinresistance encoding gene from bacteria, which is regulated by the 5′ UTRand promoter sequence for the atpA gene from C. reinhardtii and the 3′UTR sequence for the rbcL gene from C. reinhardtii. The transgenecassette is targeted to the 3HB locus of C. reinhardtii via the segmentslabeled “5′Homology” and “3′ Homology,” which are identical to sequencesof DNA flanking the 3HB locus on the 5′ and 3′ sides, respectively. AllDNA manipulations carried out in the construction of this transformingDNA were essentially as described by Sambrook et al., Molecular Cloning:A Laboratory Manual (Cold Spring Harbor Laboratory Press 1989) and Cohenet al., Meth. Enzymol. 297, 192-208, 1998.

FIG. 16A shows PCR using the gene-specific primer pair. As can be seen,multiple transformed clones are positive for insertion of theendoxylanase gene. FIG. 16B shows the PCR results using the primer pairsto differentiate homoplasmic from heteroplasmic clones. As can be seen,multiple transformed clones are either homoplasmic or heteroplasmic to adegree in favor of incorporation of the transgene. Unnumbered clonesdemonstrate the presence of wild-type psbA and, thus, were not selectedfor further analysis. Western blot analysis demonstrating proteinexpression is demonstrated in FIG. 16C.

TABLE 4 Vector Sequences SEQ ID NO. Sequence Use 19GCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTExo-β-glucanase CAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAinsertion cassetteAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCG (D1 KAN-BD01)GCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTCGAAATTAACCCTCACTAAAGGGAACAAAAGCTGGAGCTCCACCGCGGTGGCGGCCGCTCTAGCACTAGTGGATCGCCCGGGCTGCAGGAATTCcatatttagataaacgatttcaagcagcagaattagctttattagaacaaacttgtaaagaaatgaatgtaccaatgccgcgcattgtagaaaaaccagataattattatcaaattcgacgtatacgtgaattaaaacctgatttaacgattactggaatggcacatgcaaatccattagaagctcgaggtattacaacaaaatggtcagttgaatttacttttgctcaaattcatggatttactaatacacgtgaaattttagaattagtaacacagcctcttagacgcaatctaatgtcaaatcaatctgtaaatgctatttcttaatataaatcccaaaagattttttttataatactgagacttcaacacttacttgtttttattttttgtagttacaattcactcacgttaaagacattggaaaatgaggcaggacgttagtcgatatttatacactcttaagtttacttgcccaatatttatattaggacgtccccttcgggtaaataaattttagtggcagtggtaccaccactgcctattttaatactccgaagcatataaatatacttcggagtatataaatatccactaatatttatattaggcagttggcaggcaacaataaataaatttgtcccgtaaggggacgtcccgaaggggaaggggaagaaggcagttgcctcgcctatcggctaacaagttcctttggagtatataaccgcctacaggtaacttaaagaacatttgttacccgtaggggtttatacttctaattgcttcttctgaacaataaaatggtttgtgtggtctgggctaggaaacttgtaacaatgtgtagtgtcgcttccgcttcccttcgggacgtccccttcgggtaagtaaacttaggagtattaaatcgggacgtccccttcgggtaaataaatttcagtggacgtccccttacgggacgccagtagacgtcagtggcagttgcctcgcctatcggctaacaagttccttcggagtatataaatatagaatgtttacatactcctaagtttacttgcctccttcggagtatataaatatcccgaaggggaaggaggacgccagtggcagtggtaccgccactgcctgcttcctccttcggagtatgtaaaccccttcgggcaactaaagtttatcgcagtatataaatataggcagttggcaggcaactgccactgacgtcctattttaatactccgaaggaggcagttggcaggcaactgccactgacgtcccgtaagggtaaggggacgtccactggcgtcccgtaaggggaaggggacgtaggtacataaatgtgctaggtaactaacgtttgattttttgtggtataatatatgtaccatgcttttaatagaagcttgaatttataaattaaaatatttttacaatattttacggagaaattaaaactttaaaaaaattaacatATGGTACCATATCGTAAACTTGCTGTTATTAGTGCTTTCTTAGCTACTGCTCGTGCACAGTCAGCATGTACCTTACAATCTGAAACTCATCCTCCATTAACATGGCAAAAATGTTCTTCAGGAGGTACTTGTACACAACAAACTGGCTCTGTAGTAATTGATGCTAACTGGCGTTGGACACATGCCACTAATAGTTCAACTAATTGTTATGACGGTAATACTTGGTCATCAACACTTTGTCCCGATAACGAAACTTGTGCTAAAAATTGTTGTTTAGATGGTGCAGCTTACGCTTCAACTTACGGCGTTACTACATCAGGTAACTCATTATCAATTGGTTTCGTGACTCAATCAGCACAAAAAAATGTAGGCGCACGTTTATACTTAATGGCAAGTGACACAACCTATCAAGAATTTACATTATTAGGTAATGAGTTCAGTTTCGACGTAGATGTGAGTCAATTACCATGTGGTTTAAATGGTGCTCTTTTATTTCGTTTCAATGGACGCTGATGGCGGTGTAAGCAAATATCCTACTAATACAGCAGGTGCTAAATACGGAACAGGCTATTGTGATTCTCAGTGTCCTCGTGATTTAAAGTTTATTAACGGTCAAGCTAACGTGGAAGGTTGGGAACCAAGTAGTAATAATGCAAATACTGGAATTGGTGGTCACGGATCTTGTTGTTCTGAAATGGATATTTGGGAAGCTAATTCAATTAGTGAAGCATTAACTCCACATCCTTGTACTACCGTTGGCCAAGAAATTTGTGAAGGCGACGGTTGCGGTGGAACATACAGTGATAACCGTTATGGTGGTACATGTGATCCTGATGGCTGCGATTGGGACCCATATCGTTTAGGAAATACATCTTTTTATGGACCAGGAAGTTCATTCACATTAGATACAACTAAAAAGTTAACAGTTGTTACACAGTTCGAAACTAGCGGTGCTATTTAATCGTTATTACGTGCAAAATGGTGTAACTTTTCAACAACCAAATGCAGAATTAGGTTCTTATTCTGGTAACGGCCTTAATGACGATTATTGTACAGCAGAAGAAGCAGAATTTGGTGGTAGCAGCTTCTCAGATAAAGGTGGTTTAACTCAATTCAAGAAAGCAACATCAGGTGGTATGGTTTTAGTTATGTCATTATGGGATGACTATTATGCTAATATGTTATGGTTAGATAGTACATATCCTACAAACGAAACTTCAAGCACTCCTGGTGCTGTTCGTGGTTCATGTTCAACTTCAAGTGGTGTACCTGCTCAAGTTGAAAGCCAAAGTCCTAATGCAAAAGTAACTTTTAGTAATATCAAATTTGGTCCAATTGGCTCTACAGGCGATCCTTCAGGTGGTAATCCACCAGGTGGAAATCCACCTGGCACCACTACAACACGTCGTCCTGCTACTACCACAGGTTCTTCTCCTGGACCAACACAATCTCATTACGGTCAATGTGGTGGTATTGGTTATTCAGGTCCAACTGTGTGTGCATCAGGAACTACATGTCAAGTTTTAAATCCATATTATAGCCAATGTTTAGGTACCGGTGAAAACTTATACTTTCAAGGCTCAGGTGGCGGTGGAAGTGATTACAAAGATGATGATGATAAAGGAACCGGTTAATCTAGActtagcttcaactaactctagctcaaacaactaatttttttttaaactaaaataaatctggttaaccatacctggtttattttagtttagtttatacacacttttcatatatatatacttaatagctaccataggcagttggcaggacgtccccttacgggacaaatgtatttattgttgcctgccaactgcctaatataaatattagtggacgtccccttccccttacgggcaagtaaacttagggattttaatgctccgttaggaggcaaataaattttagtggcagttgcctcgcctatcggctaacaagttccttcggagtatataaatatcctgccaactgccgatatttatatactaggcagtggcggtaccactcgacGGATCCTACGTAATCGATGAATTCGATCCCATTTTTATAACTGGTCTCAAAATACCTATAAACCCATTGTTCTTCTCTTTTAGCTCTAAGAACAATCAATTTATAAATATATTTATTATTATGCTATAATATAAATACTATATAAATACATTTACCTTTTTATAAATACATTTACCTTTTTTTTAATTTGCATGATTTTAATGCTTATGCTATCTTTTTTATTTAGTCCATAAAACCTTTAAAGGACCTTTTCTTATGGGATATTTATATTTTCCTAACAAAGCAATCGGCGTCATAAACTTTAGTTGCTTACGACGCCTGTGGACGTCCCCCCCTTCCCCTTACGGGCAAGTAAACTTAGGGATTTTAATGCAATAAATAAATTTGTCCTCTTCGGGCAAATGAATTTTAGTATTTAAATATGACAAGGGTGAACCATTACTTTTGTTAACAAGTGATCTTACCACTCACTATTTTTGTTGAATTTTAAACTTATTTAAAATTCTCGAGAAAGATTTTAAAAATAAACTTTTTTAATCTTTTATTTATTTTTTCTTTTTTcgtatggaattgcccaatattattcaacaatttatcggaaacagcgttttagagccaaataaaattggtcagtcgccatcggatgtttattcttttaatcgaaataatgaaactttttttcttaagcgatctagcactttatatacagagaccacatacagtgtctctcgtgaagcgaaaatgttgagttggctctctgagaaattaaaggtgcctgaactcatcatgacttttcaggatgagcagtttgaatttatgatcactaaagcgatcaatgcaaaaccaatttcagcgctttttttaacagaccaagaattgcttgctatctataaggaggcactcaatctgttaaattcaattgctattattgattgtccatttatttcaaacattgatcatcggttaaaagagtcaaaattttttattgataaccaactccttgacgatatagatcaagatgattttgacactgaattatggggagaccataaaacttacctaagtctatggaatgagttaaccgagactcgtgttgaagaaagattggttttttctcatggcgatatcacggatagtaatatttttatagataaattcaatgaaatttattttttagaccttggtcgtgctgggttagcagatgaatttgtagatatatcctttgttgaacgttgcctaagagaggatgcatcggaggaaactgcgaaaatatttttaaagcatttaaaaaatgatagacctgacaaaaggaattattttttaaaacttgatgaattgaattgaTTCCAAGCATTATCTAAAATACTCTGCAGGCACGCTAGCTTGTACTCAAGCTCGTAACGAAGGTCGTGACCTTGCTCGTGAAGGTGGCGACGTAATTCGTTCAGCTTGTAAATGGTCTCCAGAACTTGCTGCTGCATGTGAAGTTTGGAAAGAAATTAAATTCGAATTTGATACTATTGACAAACTTTAATTTTTATTTTTCATGATGTTTATGTGAATAGCATAAACATCGTTTTTATTTTTTATGGTGTTTAGGTTAAATACCTAAACATCATTTTACATTTTTAAAATTAAGTTCTAAAGTTATCTTTTGTTTAAATTTGCCTGTGCTTTATAAATTACGATGTGCCAGAAAAATAAAATCTTAGCTTTTTATTATAGAATTTATCTTTATGTATTATATTTTATAAGTTATAATAAAAGAAATAGTAACATACTAAAGCGGATGTAGCGCGTTTATCTTAACGGAAGGAATTCGGCGCCTACGTAGGATCCgtatccatgctagcaatatctgatggtacttgcatttcataagtttggcctggaataaccaccgtttcggaagtacctgtcgctttaagttttatagctaaatctaaagtttctttaagtcttttagctgtattaaatactccacgactttcccttacgggacaataaataaatttgtccccttccccttacgtgacgtcagtggcagttgcctgccaactgcctccttcggagtattaaaatcctatatttatatactcctaagtttacttgcccaatatttatattaggcagttggcaggcaactgccactgacgtcccgaaggggaaggggaaggacgtccccttcgggtaaataaattttagtggcagtggtaccaccactgcctgcttcctccttccccttcgggcaagtaaacttagaataaaatttatttgctgcgctagcaggtttacatactcctaagtttacttgcccgaaggggaaggaggacgtccccttacgggaatataaatattagtggcagtggtacaataaataaattgtatgtaaaccccttcgggcaactaaagtttatcgcagtatataaatatagaatgtttacatactccgaaggaggacgccagtggcagtggtaccgccactgcctgtccgcagtattaacatcctattttaatactccgaaggaggcagttggcaggcaactgccactaatatttatattcccgtaaggggacgtcctaatttaatactccgaaggaggcagttggcaggcaactgccactaaaatttatttgcctcctaacggagcattaaaatcccgaaggggacgtcccgaaggggaaggggaaggaggcaactgcctgcttcctccttccccttcgggcaagtaaacttagaataaaatttatttgctgcgctagcaggtttacatactcctaagtttacttgcccgaaggggaaggaggacgtccccttacgggaatataaatattagtggcagtggtacaataaataaattgtatgtaaaccccttcgggcaactaaagtttatcgcagtatataaatatcggcagttggcaggcaactgccactaaaattcatttgcccgaaggggacgtccactaatatttatattcccgtaaggggacgtcccgaaggggaaggggacgtcctaaacggagcattaaaatccctaagtttacttgcctaggcagttggcaggatatttatatacgatattaatacttttgctactggcacactaaaatttatttgcccgtaaggggacgtccttcggtggttatataaataatcccgtagggggagggggatgtcccgtagggggaggggagtggaggctccaacggaggttggagcttctttggtttcctaggcattatttaaatattttttaaccctagcactagaactgagattccagacggcgacccgtaaagttcttcagtcccctcagctttttcacaaccaagttcgggatggattggtgtgggtccaactgagcaaagagcaccaaggttaactgcatctctgtgagatgctagttaaactaagcttagcttagctcataaacgatagttacccgcaaggggttatgtaattatattataaggtcaaaatcaaacggcctttagtatatctcggctaaagccattgctgactgtacacctgatacctatataacggcttgtctagccgcggccttagagagcactcatcttgagtttagcttcctacttagatgctttcagcagttatctatccatgcgtagctacccagcgtttcccattggaatgagaactggtacacaattggcatgtcctttcaggtcctctcgtactatgaaaggctactctcaatgctctaacgcctacaccggatatggaccaaactgtctcacgcatgaaattttaaagccgaataaaacttgcggtctttaaaactaacccctttactttcgtaaaggcatggactatgtcttcatcctgctactgttaatggcaggagtcggcgtattatactttcccactCTCGAGGGGGGGCCCGGTACCCAATTCGCCCTATAGTGAGTCGTATTACAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGCTTACAATTTAGGTG 20GCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTEndo-β-glucanaseCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGA insertioncassette AAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCG (D1KAN-BD05) GCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTCGAAATTAACCCTCACTAAAGGGAACAAAAGCTGGAGCTCCACCGCGGTGGCGGCCGCTCTAGCACTAGTGGATCGCCCGGGCTGCAGGAATTCcatatttagataaacgatttcaagcagcagaattagctttattagaacaaacttgtaaagaaatgaatgtaccaatgccgcgcattgtagaaaaaccagataattattatcaaattcgacgtatacgtgaattaaaacctgatttaacgattactggaatggcacatgcaaatccattagaagctcgaggtattacaacaaaatggtcagttgaatttacttttgctcaaattcatggatttactaatacacgtgaaattttagaattagtaacacagcctcttagacgcaatctaatgtcaaatcaatctgtaaatgctatttcttaatataaatcccaaaagattttttttataatactgagacttcaacacttacttgtttttattttttgtagttacaattcactcacgttaaagacattggaaaatgaggcaggacgttagtcgatatttatacactcttaagtttacttgcccaatatttatattaggacgtccccttcgggtaaataaattttagtggcagtggtaccaccactgcctattttaatactccgaagcatataaatatacttcggagtatataaatatccactaatatttatattaggcagttggcaggcaacaataaataaatttgtcccgtaaggggacgtcccgaaggggaaggggaagaaggcagttgcctcgcctatcggctaacaagttcctttggagtatataaccgcctacaggtaacttaaagaacatttgttacccgtaggggtttatacttctaattgcttcttctgaacaataaaatggtttgtgtggtctgggctaggaaacttgtaacaatgtgtagtgtcgcttccgcttcccttcgggacgtccccttcgggtaagtaaacttaggagtattaaatcgggacgtccccttcgggtaaataaatttcagtggacgtccccttacgggacgccagtagacgtcagtggcagttgcctcgcctatcggctaacaagttccttcggagtatataaatatagaatgtttacatactcctaagtttacttgcctccttcggagtatataaatatcccgaaggggaaggaggacgccagtggcagtggtaccgccactgcctgcttcctccttcggagtatgtaaaccccttcgggcaactaaagtttatcgcagtatataaatataggcagttggcaggcaactgccactgacgtcctattttaatactccgaaggaggcagttggcaggcaactgccactgacgtcccgtaagggtaaggggacgtccactggcgtcccgtaaggggaaggggacgtaggtacataaatgtgctaggtaactaacgtttgattttttgtggtataatatatgtaccatgcttttaatagaagcttgaatttataaattaaaatatttttacaatattttacggagaaattaaaactttaaaaaaattaacatATGGTACCAAACAAAAGCGTAGCACCATTATTACTTGCTGCATCTATCTTATATGGTGGTGCTGTTGCTCAACAGACTGTTTGGGGTCAGTGTGGTGGTATTGGTTGGTCTGGTCCTACCAATTGTGCTCCTGGCTCAGCATGTAGTACCTTAAATCCTTACTATGCTCAATGTATTCCAGGTGCAACAACTATAACAACATCAACTCGCCCTCCTTCAGGTCCAACTACAACAACTCGTGCTACTAGCACTTCTAGCAGCACACCTCCTACATCTTCTGGAGTACGTTTCGCTGGTGTTAATATTGCAGGTTTCGATTTTGGTTGTACTACCGATGGTACATGTGTTACCAGTAAAGTTTATCCCCCTTTAAAAAATTTTACTGGCTCAAACAATTATCCAGATGGCATTGGTCAAATGCAACACTTTGTAAATGAAGATGGTATGACTATTTTCCGTTTACCAGTGGGCTGGCAATACTTAGTTAACAACAATTTAGGTGGTAACTTAGATAGTACATCAATTAGTAAATATGATCAATTAGTACAAGGTTGCTTATCTTTAGGTGCCTATTGTATTGTTGATATTCATAATTATGCCCGTTGGAACGGTGGTATTATTGGTCAAGGTGGTCCAACTAATGCTCAATTTACATCATTATGGAGCCAATTAGCTTCAAAATATGCTAGTCAATCACGTGTTTGGTTCGGTATTATGAATGAACCTCACGATGTGAACATAAATACTTGGGCTGCAACTGTGCAAGAAGTAGTAACTGCTATTCGTAATGCTGGTGCAACATCACAATTCATTAGTTTACCAGGCAACGATTGGCAATCTGCCGGCGCTTTTATTTCTGACGGTAGCGCAGCTGCTCTTAGTCAAGTGACTAACCCAGACGGTAGTACCACTAACTTAATATTCGATGTACATAAATATCTTGATTCTGATAATAGCGGAACACACGCCGAATGTACCACAAATAATATTGATGGTGCTTTTAGTCCTTTAGCAACTTGGTTACGTCAAAATAATCGCCAAGCCATTTTAACTGAAACAGGTGGTGGAAACGTGCAGAGTTGTATCCAAGACATGTGTCAACAAATTCAGTACTTAAATCAAAACTCTGACGTGTACTTAGGTTATGTAGGTTGGGGTGCTGGTTCTTTTGATTCAACTTATGTATTAACCGAAACCCCTACTTCTTCTGGAAACTCATGGACAGACACTTCATTAGTAAGTAGTTGTTTAGCTCGCAAGGGTACCGGTGAAAACTTATACTTTCAAGGCTCAGGTGGCGGTGGAAGTGATTACAAAGATGATGATGATAAAGGAACCGGTTAATCTAGActtagcttcaactaactctagctcaaacaactaatttttttttaaactaaaataaatctggttaaccatacctggtttattttagtttagtttatacacacttttcatatatatatacttaatagctaccataggcagttggcaggacgtccccttacgggacaaatgtatttattgttgcctgccaactgcctaatataaatattagtggacgtccccttccccttacgggcaagtaaacttagggattttaatgctccgttaggaggcaaataaattttagtggcagttgcctcgcctatcggctaacaagttccttcggagtatataaatatcctgccaactgccgatatttatatactaggcagtggcggtaccactcgacGGATCCTACGTAATCGATGAATTCGATCCCATTTTTATAACTGGTCTCAAAATACCTATAAACCCATTGTTCTTCTCTTTTAGCTCTAAGAACAATCAATTTATAAATATATTTATTATTATGCTATAATATAAATACTATATAAATACATTTACCTTTTTATAAATACATTTACCTTTTTTTTAATTTGCATGATTTTAATGCTTATGCTATCTTTTTTATTTAGTCCATAAAACCTTTAAAGGACCTTTTCTTATGGGATATTTATATTTTCCTAACAAAGCAATCGGCGTCATAAACTTTAGTTGCTTACGACGCCTGTGGACGTCCCCCCCTTCCCCTTACGGGCAAGTAAACTTAGGGATTTTAATGCAATAAATAAATTTGTCCTCTTCGGGCAAATGAATTTTAGTATTTAAATATGACAAGGGTGAACCATTACTTTTGTTAACAAGTGATCTTACCACTCACTATTTTTGTTGAATTTTAAACTTATTTAAAATTCTCGAGAAAGATTTTAAAAATAAACTTTTTTAATCTTTTATTTATTTTTTCTTTTTTcgtatggaattgcccaatattattcaacaatttatcggaaacagcgttttagagccaaataaaattggtcagtcgccatcggatgtttattcttttaatcgaaataatgaaactttttttcttaagcgatctagcactttatatacagagaccacatacagtgtctctcgtgaagcgaaaatgttgagttggctctctgagaaattaaaggtgcctgaactcatcatgacttttcaggatgagcagtttgaatttatgatcactaaagcgatcaatgcaaaaccaatttcagcgctttttttaacagaccaagaattgcttgctatctataaggaggcactcaatctgttaaattcaattgctattattgattgtccatttatttcaaacattgatcatcggttaaaagagtcaaaattttttattgataaccaactccttgacgatatagatcaagatgattttgacactgaattatggggagaccataaaacttacctaagtctatggaatgagttaaccgagactcgtgttgaagaaagattggttttttctcatggcgatatcacggatagtaatatttttatagataaattcaatgaaatttattttttagaccttggtcgtgctgggttagcagatgaatttgtagatatatcctttgttgaacgttgcctaagagaggatgcatcggaggaaactgcgaaaatatttttaaagcatttaaaaaatgatagacctgacaaaaggaattattttttaaaacttgatgaattgaattgaTTCCAAGCATTATCTAAAATACTCTGCAGGCACGCTAGCTTGTACTCAAGCTCGTAACGAAGGTCGTGACCTTGCTCGTGAAGGTGGCGACGTAATTCGTTCAGCTTGTAAATGGTCTCCAGAACTTGCTGCTGCATGTGAAGTTTGGAAAGAAATTAAATTCGAATTTGATACTATTGACAAACTTTAATTTTTATTTTTCATGATGTTTATGTGAATAGCATAAACATCGTTTTTATTTTTTATGGTGTTTAGGTTAAATACCTAAACATCATTTTACATTTTTAAAATTAAGTTCTAAAGTTATCTTTTGTTTAAATTTGCCTGTGCTTTATAAATTACGATGTGCCAGAAAAATAAAATCTTAGCTTTTTATTATAGAATTTATCTTTATGTATTATATTTTATAAGTTATAATAAAAGAAATAGTAACATACTAAAGCGGATGTAGCGCGTTTATCTTAACGGAAGGAATTCGGCGCCTACGTAGGATCCgtatccatgctagcaatatctgatggtacttgcatttcataagtttggcctggaataaccaccgtttcggaagtacctgtcgctttaagttttatagctaaatctaaagtttctttaagtcttttagctgtattaaatactccacgactttcccttacgggacaataaataaatttgtccccttccccttacgtgacgtcagtggcagttgcctgccaactgcctccttcggagtattaaaatcctatatttatatactcctaagtttacttgcccaatatttatattaggcagttggcaggcaactgccactgacgtcccgaaggggaaggggaaggacgtccccttcgggtaaataaattttagtggcagtggtaccaccactgcctgcttcctccttccccttcgggcaagtaaacttagaataaaatttatttgctgcgctagcaggtttacatactcctaagtttacttgcccgaaggggaaggaggacgtccccttacgggaatataaatattagtggcagtggtacaataaataaattgtatgtaaaccccttcgggcaactaaagtttatcgcagtatataaatatagaatgtttacatactccgaaggaggacgccagtggcagtggtaccgccactgcctgtccgcagtattaacatcctattttaatactccgaaggaggcagttggcaggcaactgccactaatatttatattcccgtaaggggacgtcctaatttaatactccgaaggaggcagttggcaggcaactgccactaaaatttatttgcctcctaacggagcattaaaatcccgaaggggacgtcccgaaggggaaggggaaggaggcaactgcctgcttcctccttccccttcgggcaagtaaacttagaataaaatttatttgctgcgctagcaggtttacatactcctaagtttacttgcccgaaggggaaggaggacgtccccttacgggaatataaatattagtggcagtggtacaataaataaattgtatgtaaaccccttcgggcaactaaagtttatcgcagtatataaatatcggcagttggcaggcaactgccactaaaattcatttgcccgaaggggacgtccactaatatttatattcccgtaaggggacgtcccgaaggggaaggggacgtcctaaacggagcattaaaatccctaagtttacttgcctaggcagttggcaggatatttatatacgatattaatacttttgctactggcacactaaaatttatttgcccgtaaggggacgtccttcggtggttatataaataatcccgtagggggagggggatgtcccgtagggggaggggagtggaggctccaacggaggttggagcttctttggtttcctaggcattatttaaatattttttaaccctagcactagaactgagattccagacggcgacccgtaaagttcttcagtcccctcagctttttcacaaccaagttcgggatggattggtgtgggtccaactgagcaaagagcaccaaggttaactgcatctctgtgagatgctagttaaactaagcttagcttagctcataaacgatagttacccgcaaggggttatgtaattatattataaggtcaaaatcaaacggcctttagtatatctcggctaaagccattgctgactgtacacctgatacctatataacggcttgtctagccgcggccttagagagcactcatcttgagtttagcttcctacttagatgctttcagcagttatctatccatgcgtagctacccagcgtttcccattggaatgagaactggtacacaattggcatgtcctttcaggtcctctcgtactatgaaaggctactctcaatgctctaacgcctacaccggatatggaccaaactgtctcacgcatgaaattttaaagccgaataaaacttgcggtctttaaaactaacccctttactttcgtaaaggcatggactatgtcttcatcctgctactgttaatggcaggagtcggcgtattatactttcccactCTCGAGGGGGGGCCCGGTACCCAATTCGCCCTATAGTGAGTCGTATTACAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGGCTGAATGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGCTTACAATTTAGGTG 21GCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATT β-glucosidaseCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGA insertioncassette AAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCG (D1KAN-BD09) GCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTCGAAATTAACCCTCACTAAAGGGAACAAAAGCTGGAGCTCCACCGCGGTGGCGGCCGCTCTAGCACTAGTGGATCGCCCGGGCTGCAGGAATTCcatatttagataaacgatttcaagcagcagaattagctttattagaacaaacttgtaaagaaatgaatgtaccaatgccgcgcattgtagaaaaaccagataattattatcaaattcgacgtatacgtgaattaaaacctgatttaacgattactggaatggcacatgcaaatccattagaagctcgaggtattacaacaaaatggtcagttgaatttacttttgctcaaattcatggatttactaatacacgtgaaattttagaattagtaacacagcctcttagacgcaatctaatgtcaaatcaatctgtaaatgctatttcttaatataaatcccaaaagattttttttataatactgagacttcaacacttacttgtttttattttttgtagttacaattcactcagttaaagacattggaaaatgaggcaggacgttagtcgatatttatacactcttaagtttacttgcccaatatttatattaggacgtccccttcgggtaaataaattttagtggcagtggtaccaccactgcctattttaatactccgaagcatataaatatacttcggagtatataaatatccactaatatttatattaggcagttggcaggcaacaataaataaatttgtcccgtaaggggacgtcccgaaggggaaggggaagaaggcagttgcctcgcctatcggctaacaagttcctttggagtatataaccgcctacaggtaacttaaagaacatttgttacccgtaggggtttatacttctaattgcttcttctgaacaataaaatggtttgtgtggtctgggctaggaaacttgtaacaatgtgtagtgtcgcttccgcttcccttcgggacgtccccttcgggtaagtaaacttaggagtattaaatcgggacgtccccttcgggtaaataaatttcagtggacgtccccttacgggacgccagtagacgtcagtggcagttgcctcgcctatcggctaacaagttccttcggagtatataaatatagaatgtttacatactcctaagtttacttgcctccttcggagtatataaatatcccgaaggggaaggaggacgccagtggcagtggtaccgccactgcctgcttcctccttcggagtatgtaaaccccttcgggcaactaaagtttatcgcagtatataaatataggcagttggcaggcaactgccactgacgtcctattttaatactccgaaggaggcagttggcaggcaactgccactgacgtcccgtaagggtaaggggacgtccactggcgtcccgtaaggggaaggggacgtaggtacataaatgtgctaggtaactaacgtttgattttttgtggtataatatatgtaccatgcttttaatagaagcttgaatttataaattaaaatatttttacaatattttacggagaaattaaaactttaaaaaaattaacatATGGTACCATTACCAAAGGATTTCCAATGGGGTTTCGCTACCGCAGCTTATCAAATTGAAGGTGCAGTTGATCAAGATGGACGTGGACCTTCTATTTGGGACACATTCTGTGCACAACCAGGTAAAATTGCTGATGGTTCATCAGGTGTAACAGCATGTGACTCATATAATCGTACAGCTGAAGACATTGCACTTTTAAAATCTTTAGGTGCTAAATCATATCGTTTCTCTATCTCATGGTCAAGAATTATTCCTGAAGGTGGCCGTGGTGACGCAGTAAATCAAGCTGGTATTGATCACTATGTTAAATTTGTAGATGACTTATTAGACGCAGGTATTACACCTTTTATCACTTTATTTCACTGGGATTTACCTGAAGGTTTACACCAACGTTATGGTGGTCTTTTAAACCGTACAGAATTTCCTTTAGATTTCGAAAACTATGCAAGAGTTATGTTTCGTGCACTTCCCAAAGTAAGAAACTGGATTACTTTTAATGAACCTTTATGTTCTGCTATTCCTGGTTATGGTTCAGGCACCTTTGCCCCAGGCAGACAAAGTACAAGTGAGCCCTGGACAGTGGGCCATAACATTTTAGTAGCTCACGGTAGAGCTGTAAAAGCATATAGAGATGATTTCAAACCTGCTTCAGGTGATGGTCAAATAGGTATTGTGTTAAATGGTGACTTCACATATCCCTGGGATGCCGCTGATCCTGCAGATAAAGAAGCCGCTGAACGTCGCTTAGAATTTTTCACTGCTTGGTTTGCTGACCCCATCTATCTTGGTGATTATCCTGCTTCAATGCGTAAACAATTAGGTGATCGTTTACCTACTTTTACACCAGAAGAACGTGCTTTAGTTCATGGTAGTAATGACTTTTATGGTATGAACCACTATACTTCAAACTATATTCGTCACCGTAGCTCACCCGCAAGTGCTGATGACACAGTAGGTAATGTAGATGTTTTTATTTACTAATAAACAAGGTAATTGTATCGGTCCTGAAACACAGAGCCCCTGGCTTCGTCCTTGTGCAGCTGGTTTCCGTGACTTCCTTGTATGGATAAGCAAACGTTATGGTTATCCACCAATTTATGTTACAGAAAACGGAACATCAATAAAAGGTGAAAGTGACTTACCAAAGGAAAAGATTCTTGAAGATGATTTTCGTGTTAAGTATTATAACGAATACATTAGAGCTATGGTTACAGCCGTTGAATTAGATGGTGTAAATGTAAAAGGTTATTTCGCATGGTCTTTAATGGATAACTTTGAATGGGCTGATGGTTACGTTACACGTTTTGGTGTAACCTACGTTGATTACGAAAACGGCCAAAAACGTTTCCCTAAAAAGAGTGCTAAAAGTTTAAAACCTTTATTTGATGAATTAATAGCTGCTGCAGGTACCGGTGAAAACTTATACTTTCAAGGCTCAGGTGGCGGTGGAAGTGATTACAAAGATGATGATGATAAAGGAACCGGTTAATCTAGActtagcttcaactaactctagctcaaacaactaatttttttttaaactaaaataaatctggttaaccatacctggtttattttagttagtttatacacacttttcatatatatatacttaatagctaccataggcagttggcaggacgtccccttacgggacaaatgtatttattgttgcctgccaactgcctaatataaatattagtggacgtccccttccccttacgggcaagtaaacttagggattttaatgctccgttaggaggcaaataaattttagtggcagttgcctcgcctatcggctaacaagttccttcggagtatataaatatcctgccaactgccgatatttatatactaggcagtggcggtaccactcgacGGATCCTACGTAATCGATGAATTCGATCCCATTTTTATAACTGGTCTCAAAATACCTATAAACCCATTGTTCTTCTCTTTTAGCTCTAAGAACAATCAATTTATAAATATATTTATTATTATGCTATAATATAAATACTATATAAATACATTTACCTTTTTATAAATACATTTACCTTTTTTTTAATTTGCATGATTTTAATGCTTATGCTATCTTTTTTATTTAGTCCATAAAACCTTTAAAGGACCTTTTCTTATGGGATATTTATATTTTCCTAACAAAGCAATCGGCGTCATAAACTTTAGTTGCTTACGACGCCTGTGGACGTCCCCCCCTTCCCCTTACGGGCAAGTAAACTTAGGGATTTTAATGCAATAAATAAATTTGTCCTCTTCGGGCAAATGAATTTTAGTATTTAAATATGACAAGGGTGAACCATTACTTTTGTTAACAAGTGATCTTACCACTCACTATTTTTGTTGAATTTTAAACTTATTTAAAATTCTCGAGAAAGATTTTAAAAATAAACTTTTTTAATCTTTTATTTATTTTTTCTTTTTTcgtatggaattgcccaatattattcaacaatttatcggaaacagcgttttagagccaaataaaattggtcagtcgccatcggatgtttattcttttaatcgaaataatgaaactttttttcttaagcgatctagcactttatatacagagaccacatacagtgtctctcgtgaagcgaaaatgttgagttggctctctgagaaattaaaggtgcctgaactcatcatgacttttcaggatgagcagtttgaatttatgatcactaaagcgatcaatgcaaaaccaatttcagcgctttttttaacagaccaagaattgcttgctatctataaggaggcactcaatctgttaaattcaattgctattattgattgtccatttatttcaaacattgatcatcggttaaaagagtcaaaattttttattgataaccaactccttgacgatatagatcaagatgattttgacactgaattatggggagaccataaaacttacctaagtctatggaatgagttaaccgagactcgtgttgaagaaagattggttttttctcatggcgatatcacggatagtaatatttttatagataaattcaatgaaatttattttttagaccttggtcgtgctgggttagcagatgaatttgtagatatatcctttgttgaacgttgcctaagagaggatgcatcggaggaaactgcgaaaatatttttaaagcatttaaaaaatgatagacctgacaaaaggaattattttttaaaacttgatgaattgaattgaTTCCAAGCATTATCTAAAATACTCTGCAGGCACGCTAGCTTGTACTCAAGCTCGTAACGAAGGTCGTGACCTTGCTCGTGAAGGTGGCGACGTAATTCGTTCAGCTTGTAAATGGTCTCCAGAACTTGCTGCTGCATGTGAAGTTTGGAAAGAAATTAAATTCGAATTTGATACTATTGACAAACTTTAATTTTTATTTTTCATGATGTTTATGTGAATAGCATAAACATCGTTTTTATTTTTTATGGTGTTTAGGTTAAATACCTAAACATCATTTTACATTTTTAAAATTAAGTTCTAAAGTTATCTTTTGTTTAAATTTGCCTGTGCTTTATAAATTACGATGTGCCAGAAAAATAAAATCTTAGCTTTTTATTATAGAATTTATCTTTATGTATTATATTTTATAAGTTATAATAAAAGAAATAGTAACATACTAAAGCGGATGTAGCGCGTTTATCTTAACGGAAGGAATTCGGCGCCTACGTAGGATCCgtatccatgctagcaatatctgatggtacttgcatttcataagtttggcctggaataaccaccgtttcggaagtacctgtcgcgtttaagttttatagctaaatctaaagtttctttaagtcttttagctgtattaaatactccacgactttccctacgggacaataaataaatttgtccccttccccttacgtgacgtcagtggcagttgcctgccaactgcctccttcggagtattaaaatcctatatttatatactcctaagtttacttgcccaatatttatattaggcagttggcaggcaactgccactgacgtcccgaaggggaaggggaaggacgtccccttcgggtaaataaattttagtggcagtggtaccaccactgcctgcttcctccttccccttcgggcaagtaaacttagaataaaatttatttgctgcgctagcaggtttacatactcctaagtttacttgcccgaaggggaaggaggacgtccccttacgggaatataaatattagtggcagtggtacaataaataaattgtatgtaaaccccttcgggcaactaaagtttatcgcagtatataaatatagaatgtttacatactccgaaggaggacgccagtggcagtggtaccgccactgcctgtccgcagtattaacatcctattttaatactccgaaggaggcagttggcaggcaactgccactaatatttatattcccgtaaggggacgtcctaatttaatactccgaaggaggcagttggcaggcaactgccactaaaatttatttgcctcctaacggagcattaaaatcccgaaggggacgtcccgaaggggaaggggaaggaggcaactgcctgcttcctccttccccttcgggcaagtaaacttagaataaaatttatttgctgcgctagcaggtttacatactcctaagtttacttgcccgaaggggaaggaggacgtccccttacgggaatataaatattagtggcagtggtacaataaataaattgtatgtaaaccccttcgggcaactaaagtttatcgcagtatataaatatcggcagttggcaggcaactgccactaaaattcatttgcccgaaggggacgtccactaatatttatattcccgtaaggggacgtcccgaaggggaaggggacgtcctaaacggagcattaaaatccctaagtttacttgcctaggcagttggcaggatatttatatacgatattaatacttttgctactggcacactaaaatttatttgcccgtaaggggacgtccttcggtggttatataaataatcccgtagggggagggggatgtcccgtagggggaggggagtggaggctccaacggaggttggagcttctttggtttcctaggcattatttaaatattttttaaccctagcactagaactgagattccagacggcgacccgtaaagttcttcagtcccctcagctttttcacaaccaagttcgggatggattggtgtgggtccaactgagcaaagagcaccaaggttaactgcatctctgtgagatgctagttaaactaagcttagcttagctcataaacgatagttacccgcaaggggttatgtaattatattataaggtcaaaatcaaacggcctttagtatatctcggctaaagccattgctgactgtacacctgatacctatataacggcttgtctagccgcggccttagagagcactcatcttgagtttagcttcctacttagatgctttcagcagttatctatccatgcgtagctacccagcgtttcccattggaatgagaactggtacacaattggcatgtcctttcaggtcctctcgtactatgaaaggctactctcaatgctctaacgcctacaccggatatggaccaaactgtctcacgcatgaaattttaaagccgaataaaacttgcggtctttaaaactaacccctttactttcgtaaaggcatggactatgtcttcatcctgctactgttaatggcaggagtcggcgtattatactttcccactCTCGAGGGGGGGCCCGGTACCCAATTCGCCCTATAGTGAGTCGTATTACAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGCTTACAATTTA GGTG 22GCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATT Endo-xylanaseCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGA insertioncassette AAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCG (D1KAN-BD11) GCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTCGAAATTAACCCTCACTAAAGGGAACAAAAGCTGGAGCTCCACCGCGGTGGCGGCCGCTCTAGCACTAGTGGATCGCCCGGGCTGCAGGAATTCcatatttagataaacgatttcaagcagcagaattagctttattagaacaaacttgtaaagaaatgaatgtaccaatgccgcgcattgtagaaaaaccagataattattatcaaattcgacgtatacgtgaattaaaacctgatttaacgattactggaatggcacatgcaaatccattagaagctcgaggtattacaacaaaatggtcagttgaatttacttttgctcaaattcatggatttactaatacacgtgaaattttagaattagtaacacagcctcttagacgcaatctaatgtcaaatcaatctgtaaatgctatttcttaatataaatcccaaaagattttttttataatactgagacttcaacacttacttgtttttattttttgtagttacaattcactcacgttaaagacattggaaaatgaggcaggacgttagtcgatatttatacactcttaagtttacttgcccaatatttatattaggacgtccccttcgggtaaataaattttagtggcagtggtaccaccactgcctattttaatactccgaagcatataaatatacttcggagtatataaatatccactaatatttatattaggcagttggcaggcaacaataaataaatttgtcccgtaaggggacgtcccgaaggggaaggggaagaaggcagttgcctcgcctatcggctaacaagttcctttggagtatataaccgcctacaggtaacttaaagaacatttgttacccgtaggggtttatacttctaattgcttcttctgaacaataaaatggtttgtgtggtctgggctaggaaacttgtaacaatgtgtagtgtcgcttccgcttcccttcgggacgtccccttcgggtaagtaaacttaggagtattaaatcgggacgtccccttcgggtaaataaatttcagtggacgtccccttacgggacgccagtagacgtcagtggcagttgcctcgcctatcggctaacaagttccttcggagtatataaatatagaatgtttacatactcctaagtttacttgcctccttcggagtatataaatatcccgaaggggaaggaggacgccagtggcagtggtaccgccactgcctgcttcctccttcggagtatgtaaaccccttcgggcaactaaagtttatcgcagtatataaatataggcagttggcaggcaactgccactgacgtcctattttaatactccgaaggaggcagttggcaggcaactgccactgacgtcccgtaagggtaaggggacgtccactggcgtcccgtaaggggaaggggacgtaggtacataaatgtgctaggtaactaacgtttgattttttgtggtataatatatgtaccatgcttttaatagaagcttgaatttataaattaaaatatttttacaatattttacggagaaattaaaactttaaaaaaattaacatATGGTACCAGTATCTTTCACAAGTCTTTTAGCAGCATCTCCACCTTCACGTGCAAGTTGCCGTCCAGCTGCTGAAGTGGAATCAGTTGCAGTAGAAAAACGTCAAACAATTCAACCAGGTACAGGTTACAATAACGGTTACTTTTATTCTTACTGGAATGATGGACACGGTGGTGTTACATATACTAATGGACCTGGTGGTCAATTTAGTGTAAATTGGAGTAACTCAGGCAATTTTGTTGGAGGAAAAGGTTGGCAACCTGGTACAAAGAATAAGGTAATCAATTTCTCTGGTAGTTACAACCCTAATGGTAATTCTTATTTAAGTGTATACGGTTGGAGCCGTAACCCATTAATTGAATATTATATTGTAGAGAACTTTGGTACATACAACCCTTCAACAGGTGCTACTAAATTAGGTGAAGTTACTTCAGATGGATCAGTTTATGATATTTATCGTACTCAACGCGTAAATCAACCATCTATAATTGGAACTGCCACTTTCTACCAATACTGGAGTGTAAGACGTAATCATCGTTCAAGTGGTAGTGTTAATACAGCAAACCACTTTAATGCATGGGCTCAACAAGGTTTAACATTAGGTACAATGGACTATCAAATTGTAGCTGTTGAAGGTTATTTTTCATCAGGTAGTGCTTCTATCACTGTTAGCGGTACCGGTGAAAACTTATACTTTCAAGGCTCAGGTGGCGGTGGAAGTGATTACAAAGATGATGATGATAAAGGAACCGGTTAATCTAGActtagcttcaactaactctagctcaaacaactaatttttttttaaactaaaataaatctggttaaccatacctggtttattttagtttagtttatacacacttttcatatatatatacttaatagctaccataggcagttggcaggacgtccccttacgggacaaatgtatttattgttgcctgccaactgcctaatataaatattagtggacgtccccttccccttacgggcaagtaaacttagggattttaatgctccgttaggaggcaaataaattttagtggcagttgcctcgcctatcggctaacaagttccttcggagtatataaatatcctgccaactgccgatatttatatactaggcagtggcggtaccactcgacGGATCCTACGTAATCGATGAATTCGATCCCATTTTTATAACTGGTCTCAAAATACCTATAAACCCATTGTTCTTCTCTTTTAGCTCTAAGAACAATCAATTTATAAATATATTTATTATTATGCTATAATATAAATACTATATAAATACATTTACCTTTTTATAAATACATTTACCTTTTTTTTAATTTGCATGATTTTAATGCTTATGCTATCTTTTTTATTTAGTCCATAAAACCTTTAAAGGACCTTTTCTTATGGGATATTTATATTTTCCTAACAAAGCAATCGGCGTCATAAACTTTAGTTGCTTACGACGCCTGTGGACGTCCCCCCCTTCCCCTTACGGGCAAGTAAACTTAGGGATTTTAATGCAATAAATAAATTTGTCCTCTTCGGGCAAATGAATTTTAGTATTTAAATATGACAAGGGTGAACCATTACTTTTGTTAACAAGTGATCTTACCACTCACTATTTTTGTTGAATTTTAAACTTATTTAAAATTCTCGAGAAAGATTTTAAAAATAAACTTTTTTAATCTTTTATTTATTTTTTCTTTTTTcgtatggaattgcccaatattattcaacaatttatcggaaacagcgttttagagccaaataaaattggtcagtcgccatcggatgtttattcttttaatcgaaataatgaaactttttttcttaagcgatctagcactttatatacagagaccacatacagtgtctctcgtgaagcgaaaatgttgagttggctctctgagaaattaaaggtgcctgaactcatcatgacttttcaggatgagcagtttgaatttatgatcactaaagcgatcaatgcaaaaccaatttcagcgctttttttaacagaccaagaattgcttgctatctataaggaggcactcaatctgttaaattcaattgctattattgattgtccatttatttcaaacattgatcatcggttaaaagagtcaaaattttttattgataaccaactccttgacgatatagatcaagatgattttgacactgaattatggggagaccataaaacttacctaagtctatggaatgagttaaccgagactcgtgttgaagaaagattggttttttctcatggcgatatcacggatagtaatatttttatagataaattcaatgaaatttattttttagaccttggtcgtgctgggttagcagatgaatttgtagatatatcctttgttgaacgttgcctaagagaggatgcatcggaggaaactgcgaaaatatttttaaagcatttaaaaaatgatagacctgacaaaaggaattattttttaaaacttgatgaattgaattgaTTCCAAGCATTATCTAAAATACTCTGCAGGCACGCTAGCTTGTACTCAAGCTCGTAACGAAGGTCGTGACCTTGCTCGTGAAGGTGGCGACGTAATTCGTTCAGCTTGTAAATGGTCTCCAGAACTTGCTGCTGCATGTGAAGTTTGGAAAGAAATTAAATTCGAATTTGATACTATTGACAAACTTTAATTTTTATTTTTCATGATGTTTATGTGAATAGCATAAACATCGTTTTTATTTTTTATGGTGTTTAGGTTAAATACCTAAACATCATTTTACATTTTTAAAATTAAGTTCTAAAGTTATCTTTTGTTTAAATTTGCCTGTGCTTTATAAATTACGATGTGCCAGAAAAATAAAATCTTAGCTTTTTATTATAGAATTTATCTTTATGTATTATATTTTATAAGTTATAATAAAAGAAATAGTAACATACTAAAGCGGATGTAGCGCGTTTATCTTAACGGAAGGAATTCGGCGCCTACGTAGGATCCgtatccatgctagcaatatctgatggtacttgcatttcataagtttggcctggaataaccaccgtttcggaagtacctgtcgctttaagttttatagctaaatctaaagtttctttaagtcttttagctgtattaaatactccacgactttcccttacgggacaataaataaatttgtccccttccccttacgtgacgtcagtggcagttgcctgccaactgcctccttcggagtattaaaatcctatatttatatactcctaagtttacttgcccaatatttatattaggcagttggcaggcaactgccactgacgtcccgaaggggaaggggaaggacgtccccttcgggtaaataaattttagtggcagtggtaccaccactgcctgcttcctccttccccttcgggcaagtaaacttagaataaaatttatttgctgcgctagcaggtttacatactcctaagtttacttgcccgaaggggaaggaggacgtccccttacgggaatataaatattagtggcagtggtacaataaataaattgtatgtaaaccccttcgggcaactaaagtttatcgcagtatataaatatagaatgtttacatactccgaaggaggacgccagtggcagtggtaccgccactgcctgtccgcagtattaacatcctattttaatactccgaaggaggcagttggcaggcaactgccactaatatttatattcccgtaaggggacgtcctaatttaatactccgaaggaggcagttggcaggcaactgccactaaaatttatttgcctcctaacggagcattaaaatcccgaaggggacgtcccgaaggggaaggggaaggaggcaactgcctgcttcctccttccccttcgggcaagtaaacttagaataaaatttatttgctgcgctagcaggtttacatactcctaagtttacttgcccgaaggggaaggaggacgtccccttacgggaatataaatattagtggcagtggtacaataaataaattgtatgtaaaccccttcgggcaactaaagtttatcgcagtatataaatatcggcagttggcaggcaactgccactaaaattcatttgcccgaaggggacgtccactaatatttatattcccgtaaggggacgtcccgaaggggaaggggacgtcctaaacggagcattaaaatccctaagtttacttgcctaggcagttggcaggatatttatatacgatattaatacttttgctactggcacactaaaatttatttgcccgtaaggggacgtccttcggtggttatataaataatcccgtagggggagggggatgtcccgtagggggaggggagtggaggctccaacggaggttggagcttctttggtttcctaggcattatttaaatattttttaaccctagcactagaactgagattccagacggcgacccgtaaagttcttcagtcccctcagctttttcacaaccaagttcgggatggattggtgtgggtccaactgagcaaagagcaccaaggttaactgcatctctgtgagatgctagttaaactaagcttagcttagctcataaacgatagttacccgcaaggggttatgtaattatattataaggtcaaaatcaaacggcctttagtatatctcggctaaagccattgctgactgtacacctgatacctatataacggcttgtctagccgcggccttagagagcactcatcttgagtttagcttcctacttagatgctttcagcagttatctatccatgcgtagctacccagcgtttcccattggaatgagaactggtacacaattggcatgtcctttcaggtcctctcgtactatgaaaggctactctcaatgctctaacgcctacaccggatatggaccaaactgtctcacgcatgaaattttaaagccgaataaaacttgcggtctttaaaactaacccctttactttcgtaaaggcatggactatgtcttcatcctgctactgttaatggcaggagtcggcgtattatactttcccactCTCGAGGGGGGGCCCGGTACCCAATTCGCCCTATAGTGAGTCGTATTACAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGCTTACAATT TAGGTG 23GTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGCCCCGACACCC β-glucosidaseinsertion GCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAcassette GACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCAC (3HBKAN-rbcL-BD09) CGAAACGCGCGAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCaagctcgcggccgcagtactCTGCAGATTTTATGCAAAATTAAAGTCTTGTGACAACAGCTTTCTCCTTAAGTGCAAATATCGCCCATTCTTTCCTCTTTTCGTATATAAATGCTGTAATAGTAGGATGTCGTACCCGTAAAGGTACGACATTGAATATTAATATACTCCTAAGTTTACTTTCCCAATATTTATATTAGGACGTCCCCTTCGGGTAAATAAATTTTAGTGGCAGTGGTACCGCCACTCCCTATTTTAATACTGCGAAGGAGGCAGTTGGCAGGCAACTCGTCGTTCGCAGTATATAAATATCCACTAATATTTATATTCCCGTAAGGGGACGTCCCGAAGGGGAAGGGGAAAGAAGCAGTCGCCTCCTTGCGAAAAGGTTTACTTGCCCGACCAGTGAAAAGCATGCTGTAAGATATAAATCTACCCTGAAAGGGATGCATTTCACCATAATACTATACAAATGGTGTTACCCTTTGAGGATCATAACGGTGCTACTGGAATATATGGTCTCTTCATGGATAGACGATAGCCATTTATTTACCCATTAAGGGGACATTAGTGGCCTGTCACTGCTCCTTACGAGACGCCAGTGGACGTTCGTCCTAGAAAATTTATGCGCTGCCTAGAAGCCCCAAAAGGGAAGTTTACTGACTCGTTAGAGCGTGCGCTAACAGGTTTAAATACTTCAATATGTATATTAGGACGCCGGTGGCAGTGGTACCGCCACTGCCACCGTCGGAGGACGTCCCTTACGGTATATTATATACTAGGATTTTAATACTCCGAAGGAGGCAGTGGCGGTACCACTGCCACTAATATTTATATTCCCGTAAGGGACGTCCTCCTTCGGAGTATGTAAACATTCTAAGTTTACTTGCCCAATATTTATATTAGGCAGTTGGCAGGCAACTGCTAGCTCTCCTCCTTCGGAGTATGTAAACATCGCAGTATATAAATATCCACTAATATTTATATTCCCGTAAGGGGACGTCCCGAAGGGGAAGGGGAAGGACGTCAGTGGCAGTTGCCTGCCAACTGCCTAGGCAAGTAAACTTAGGAGTATATAAATATAGGCAGTCGCGGTACCACTGCCACTGACGTCCTGCCAACTGCCTAGGCAAGTAAACTTAAGTGGCACTAAAATGCATTTGCCCGAAGGGGAAGGAGGACGCCAGTGGCAGTGGTACCGCCACTGCCTCCTTCGGAGTATTAAAATCCTAGTATGTAAATCTGCTAGCGCAGGAAATAAATTTTATTCTATTTATATACTCCGTTAGGAGGTAAGTAAACCCCTTCCCCTTCGGGACGTCAGTGCAGTTGCCTGCCAACTGCCTAATATAAATATTAGACCACTAAAGTTTGGCAACTGCCAACTGTTGTCCTTCGGAGGAAAAAAAATGGTTAACTCGCAAGCAGTTAACATAACTAAAGTTTGTTACTTTACCGAAGACGTTTACCCTTTCTCGGTTAAGGAGACGGAGACAGTTGCACTGTGACTGCCTAGTATAGCAATTTTGTTTTTGTTTATATGCTCGACAAAATGACTTTCATAAAAATATAAAGTAGTTAGCTAGTTATTTTATATCACTATAACTAGGGTTCTCAGAGGCACCGAAGTCACTTGTAAAAATAGTACTTTTTAACTTGTTTAATCTTCGTGTTCTTCAAAAGGATCACGTAATTTTTTTGAAGGTGGACCAAAACTAACATAAACTGAATAGCCAGTTACACTTAACAGAAGAAACCATAAAAAAAAGGTAAAGAAAAAAGCTGGACTTTCCATAGCTCATTTAATAATAAAATTATTCTCTTTTCAACATATCTCTTAGATAGTTCAAAAGACTTGACGACTGTGTCCCACATTTTTAAACAAAATTAATCTACTCAAAATTTTGCCCTGAGAAAGAATAACTTACTTCGTTTTTGCAGTAGCCATTCATGTCACTTTGAAACTGTCCTTACAAAGTTAAACATTAATTAAAAATTATTTAATTTTTATATAACAAATATTATATTAAATAAAAAATGAACAAAGAACTTCTAAGATCGTCTTTAGTGAGTAATTAAAGAGTTTTACTTACCAGACAAGGCAGTTTTTTCATTCTTTTAAAGCAGGCAGTTCTGAAGGGGAAAAGGGACTGCCTACTGCGGTCCTAGGTAAATACATTTTTATGCAATTTATTTCTTGTGCTAGTAGGTTTCTATACTCACAAGAAGCAACCCCTTGACGAGAGAACGTTATCCTCAGAGTATTTATAATCCTGAGAGGGAATGCACTGAAGAATATTTTCCTTATTTTTTACAGAAAGTAAATAAAATAGCGCTAATAACGCTTAATTCATTTAATCAATTATGGCAACAGGAACTTCTAAAGCTAAACCATCAAAAGTAAATTCAGACTTCCAAGAACCTGGTTTAGTTACACCATTAGGTACTTTATTACGTCCACTTAACTCAGAAGCAGGTAAAGTATTACCAGGCTGGGGTACAACTGTTTTAATGGCTGTATTTATCCTTTTATTTGCAGCATTCTTATTAATCATTTTAGAAATTTACAACAGTTCTTTAATTTTAGATGACGTTTCTATGAGTTGGGAAACTTTAGCTAAAGTTTCTTAATTTTATTTAACACAAACATAAAATATAAAACTGTTTGTTAAGGCTAGCTGCTAAGTCTTCTTTTCGCTAAGGTAAACTAAGCAACTCAACCATATTTATATTCGGCAGTGGCACCGCCAACTGCCACTGGCCTTCCGTTAAGATAAACGCGTggatctcacgtgactagtcacctagtgtcgagtggtaccgccactgcctagtatataaatatcggcagttggcaggatatttatatactccgaaggaacttgttagccgataggcgaggcaactgccactaaaatttatttgcctcctaacggagcattaaaatccctaagtttacttgcccgtaaggggaaggggacgtccactaatatttatattaggcagttggcaggcaacaataaatacatttgtcccgtaaggggacgtcctgccaactgcctatggtagctattaagtatatatatatgaaaagtgtgtataaactaaactaaaataaaccaggtatggttaaccagatttattttagtttaaaaaaaaattagttgtttgagctagagttagttgaagctaagtctagaTTAACCGGTTCCTTTATCATCATCATCTTTGTAATCACTTCCACCGCCACCTGAGCCTTGAAAGTATAAGTTTTCACCGGTACCTGCAGCAGCTATTAATTCATCAAATAAAGGTTTTAAACTTTTAGCACTCTTTTTAGGGAAACGTTTTTGGCCGTTTTCGTAATCAACGTAGGTTACACCAAAACGTGTAACGTAACCATCAGCCCATTCAAAGTTATCCATTAAAGACCATGCGAAATAACCTTTTACATTTACACCATCTAATTCAACGGCTGTAACCATAGCTCTAATGTATTCGTTATAATACTTAACACGAAAATCATCTTCAAGAATCTTTTCCTTTGGTAAGTCACTTTCACCTTTTATTGATGTTCCGTTTTCTGTAACATAAATTGGTGGATAACCATAACGTTTGCTTATCCATACAAGGAAGTCACGGAAACCAGCTGCACAAGGACGAAGCCAGGGGCTCTGTGTTTCAGGACCGATACAATTACCTTGTTTATTAGTAAATAAAACATCTACATTACCTACTGTGTCATCAGCACTTGCGGGTGAGCTACGGTGACGAATATAGTTTGAAGTATAGTGGTTCATACCATAAAAGTCATTACTACCATGAACTAAAGCACGTTCTTCTGGTGTAAAAGTAGGTAAACGATCACCTAATTGTTTACGCATTGAAGCAGGATAATCACCAAGATAGATGGGGTCAGCAAACCAAGCAGTGAAAAATTCTAAGCGACGTTCAGCGGCTTCTTTATCTGCAGGATCAGCGGCATCCCAGGGATATGTGAAGTCACCATTTAACACAATACCTATTTGACCATCACCTGAAGCAGGTTTGAAATCATCTCTATATGCTTTTACAGCTCTACCGTGAGCTACTAAAATGTTATGGCCCACTGTCCAGGGCTCACTTGTACTTTGTCTGCCTGGGGCAAAGGTGCCTGAACCATAACCAGGAATAGCAGAACATAAAGGTTCATTAAAAGTAATCCAGTTTCTTACTTTGGGAAGTGCACGAAACATAACTCTTGCATAGTTTTCGAAATCTAAAGGAAATTCTGTACGGTTTAAAAGACCACCATAACGTTGGTGTAAACCTTCAGGTAAATCCCAGTGAAATAAAGTGATAAAAGGTGTAATACCTGCGTCTAATAAGTCATCTACAAATTTAACATAGTGATCAATACCAGCTTGATTTACTGCGTCACCACGGCCACCTTCAGGAATAATTCTTGACCATGAGATAGAGAAACGATATGATTTAGCACCTAAAGATTTTAAAAGTGCAATGTCTTCAGCTGTACGATTATATGAGTCACATGCTGTTACACCTGATGAACCATCAGCAATTTTACCTGGTTGTGCACAGAATGTGTCCCAAATAGAAGGTCCACGTCCATCTTGATCAACTGCACCTTCAATTTGATAAGCTGCGGTAGCGAAACCCCATTGGAAATCCTTTGGTAATGGTACCATatgcactttgcattacctccgtacaaattattttgatttctataaagttttgcttaaataaaaatttttaatttttaacgtccacccatataaataataaatatggtgaaacctttaacaacaaaaatcctcttgtaccatattaatccaaaagaattaaggacaaaagcttatctccaacatttttaaaacacagagtaaaaataatgttgtttttaagaatagaattttataacttgtattttaaatatgatctaatttatttgtgctaaaaattgcagttggaaagtaattttaaaaataatttagatcatatttattaaataaagttgatttaaaacaacttaatcgtttttaattgttaattaaaaacataattttaaatctttttatatttaaattaccttatatactactagtgatatctacgtaatcgatgaattcgatcccatttttataactggatctcaaaatacctataaacccattgttcttctcttttagctctaagaacaatcaatttataaatatatttattattatgctataatataaatactatataaatacatttacctttttataaatacatttaccttttttttaatttgcatgattttaatgcttatgctatcttttttatttagtccataaaacctttaaaggaccttttcttatgggatatttatattttcctaacaaagcaatcggcgtcataaactttagttgcttacgacgcctgtggacgtcccccccttccccttacgggcaagtaaacttagggattttaatgcaataaataaatttgtcctcttcgggcaaatgaattttagtatttaaatatgacaagggtgaaccattacttttgttaacaagtgatcttaccactcactatttttgttgaattttaaacttatttaaaattctcgagaaagattttaaaaataaacttttttaatcttttatttattttttcttttttCGTATGGAATTGCCCAATATTATTCAACAATTTATCGGAAACAGCGTTTTAGAGCCAAATAAAATTGGTCAGTCGCCATCGGATGTTTATTCTTTTAATCGAAATAATGAAACTTTTTTTCTTAAGCGATCTAGCACTTTATATACAGAGACCACATACAGTGTCTCTCGTGAAGCGAAAATGTTGAGTTGGCTCTCTGAGAAATTAAAGGTGCCTGAACTCATCATGACTTTTCAGGATGAGCAGTTTGAATTTATGATCACTAAAGCGATCAATGCAAAACCAATTTCAGCGCTTTTTTTAACAGACCAAGAATTGCTTGCTATCTATAAGGAGGCACTCAATCTGTTAAATTCAATTGCTATTATTGATTGTCCATTTATTTCAAACATTGATCATCGGTTAAAAGAGTCAAAATTTTTTATTGATAACCAACTCCTTGACGATATAGATCAAGATGATTTTGACACTGAATTATGGGGAGACCATAAAACTTACCTAAGTCTATGGAATGAGTTAACCGAGACTCGTGTTGAAGAAAGATTGGTTTTTTCTCATGGCGATATCACGGATAGTAATATTTTTATAGATAAATTCAATGAAATTTATTTTTTAGACCTTGGTCGTGCTGGGTTAGCAGATGAATTTGTAGATATATCCTTTGTTGAACGTTGCCTAAGAGAGGATGCATCGGAGGAAACTGCGAAAATATTTTTAAAGCATTTAAAAAATGATAGACCTGACAAAAGGAATTATTTTTTAAAACTTGATGAATTGAATTGAttccaagcattatctaaaatactctgcaggcacgctagcttgtactcaagctcgtaacgaaggtcgtgaccttgctcgtgaaggtggcgacgtaattcgttcagcttgtaaatggtctccagaacttgctgctgcatgtgaagtttggaaagaaattaaattcgaatttgatactattgacaaactttaatttttatttttcatgatgtttatgtgaatagcataaacatcgtttttatttttatggtgtttaggttaaatacctaaacatcattttacatttttaaaattaagttctaaagttatcttttgtttaaatttgcctgtctttataaattacgatgtgccagaaaaataaaatcttagctttttattatagaatttatctttatgtattatattttataagttataataaaagaaatagtaacatactaaagcggatgtagcgcgtttatcttaacggaaggaattcggcgcctacgtacccgggtcgcgaggatccACGCGTTAATAGCTCACTTTTCTTTAAATTTAATTTTTAATTTAAAGGTGTAAGCAAATTGCCTGACGAGAGATCCACTTAAAGGATGACAGTGGCGGGCTACTGCCTACTTCCCTCCGGGATAAAATTTATTTGAAAAACGTTAGTTACTTCCTAACGGAGCATTGACATCCCCATATTTATATTAGGACGTCCCCTTCGGGTAAATAAATTTTAGTGGACGTCCCCTTCGGGCAAATAAATTTTAGTGGACAATAAATAAATTTGTTGCCTGCCAACTGCCTAGGCAAGTAAACTTGGGAGTATTAAAATAGGACGTCAGTGGCAGTTGCCTGCCAACTGCCTATATTTATATACTGCGAAGCAGGCAGTGGCGGTACCACTGCCACTGGCGTCCTAATATAAATATTGGGCAACTAAAGTTTATAGCAGTATTAACATCCTATATTTATATACTCCGAAGGAACTTGTTAGCCGATAGGCGAGGCAACAAATTTATTTATTGTCCCGTAAAAGGATGCCTCCAGCATCGAAGGGGAAGGGGACGTCCTAGGCCATAAAACTAAAGGGAAATCCATAGTAACTGATGTTATAAATTTATAGACTCCAAAAAACAGCTGCGTTATAAATAACTTCTGTTAAATATGGCCAAGGGGACAGGGGCACTTTCAACTAAGTGTACATTAAAAATTGACAATTCAATTTTTTTTAATTATAATATATATTTAGTAAAATATAACAAAAAGCCCCCATCGTCTAGgtagaattccagctggcggccgccctatg 24agatctcgatcccgcgaaattaatacgactcactataggggaattgtgagcggataacaattcccctctagaaataattttgtttaactttaagaExo-β- aggagatataCATATGGTACCATATCGTAAACTTGCTGTTATTAGTGCTTTCTTAGCTACTglucanase GCTCGTGCACAGTCAGCATGTACCTTACAATCTGAAACTCATCCTCCATTAACATGGinsertion cassetteCAAAAATGTTCTTCAGGAGGTACTTGTACACAACAAACTGGCTCTGTAGTAATTGAT (pET-21a-BD01)GCTAACTGGCGTTGGACACATGCCACTAATAGTTCAACTAATTGTTATGACGGTAATACTTGGTCATCAACACTTTGTCCCGATAACGAAACTTGTGCTAAAAATTGTTGTTTAGATGGTGCAGCTTACGCTTCAACTTACGGCGTTACTACATCAGGTAACTCATTATCAATTGGTTTCGTGACTCAATCAGCACAAAAAAATGTAGGCGCACGTTTATACTTAATGGCAAGTGACACAACCTATCAAGAATTTACATTATTAGGTAATGAGTTCAGTTTCGACGTAGATGTGAGTCAATTACCATGTGGTTTAAATGGTGCTCTTTATTTCGTTTCAATGGACGCTGATGGCGGTGTAAGCAAATATCCTACTAATACAGCAGGTGCTAAATACGGAACAGGCTATTGTGATTCTCAGTGTCCTCGTGATTTAAAGTTTATTAACGGTCAAGCTAACGTGGAAGGTTGGGAACCAAGTAGTAATAATGCAAATACTGGAATTGGTGGTCACGGATCTTGTTGTTCTGAAATGGATATTTGGGAAGCTAATTCAATTAGTGAAGCATTAACTCCACATCCTTGTACTACCGTTGGCCAAGAAATTTGTGAAGGCGACGGTTGCGGTGGAACATACAGTGATAACCGTTATGGTGGTACATGTGATCCTGATGGCTGCGATTGGGACCCATATCGTTTAGGAAATACATCTTTTTATGGACCAGGAAGTTCATTCACATTAGATACAACTAAAAAGTTAACAGTTGTTACACAGTTCGAAACTAGCGGTGCTATTAATCGTTATTACGTGCAAAATGGTGTAACTTTTCAACAACCAAATGCAGAATTAGGTTCTTATTCTGGTAACGGCCTTAATGACGATTATTGTACAGCAGAAGAAGCAGAATTTGGTGGTAGCAGCTTCTCAGATAAAGGTGGTTTAACTCAATTCAAGAAAGCAACATCAGGTGGTATGGTTTTAGTTATGTCATTATGGGATGACTATTATGCTAATATGTTATGGTTAGATAGTACATATCCTACAAACGAAACTTCAAGCACTCCTGGTGCTGTTCGTGGTTCATGTTCAACTTCAAGTGGTGTACCTGCTCAAGTTGAAAGCCAAAGTCCTAATGCAAAAGTAACTTTTAGTAATATCAAATTTGGTCCAATTGGCTCTACAGGCGATCCTTCAGGTGGTAATCCACCAGGTGGAAATCCACCTGGCACCACTACAACACGTCGTCCTGCTACTACCACAGGTTCTTCTCCTGGACCAACACAATCTCATTACGGTCAATGTGGTGGTATTGGTTATTCAGGTCCAACTGTGTGTGCATCAGGAACTACATGTCAAGTTTTAAATCCATATTATAGCCAATGTTTAGGTACCGGTGAAAACTTATACTTTCAAGGCTCAGGTGGCGGTGGAAGTGATTACAAAGATGATGATGATAAAGGAACCGGTTAATCTAGACTCGAGcaccaccaccaccaccactgagatccggctgctaacaaagcccgaaaggaagctgagttggctgctgccaccgctgagcaataactagcataaccccttggggcctctaaacgggtcttgaggggttttttgctgaaaggaggaactatatccggattggcgaatgggacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgtttacaatttcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgcagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcctgatgcggtattttctccttacgcatctgtgcggtatttcacaccgcatatatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagtatacactccgctatcgctacgtgactgggtcatggctgcgccccgacacccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttacagacaagctgtgaccgtctccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgaggcagctgcggtaaagctcatcagcgtggtcgtgaagcgattcacagatgtctgcctgttcatccgcgtccagctcgttgagtttctccagaagcgttaatgtctggcttctgataaagcgggccatgttaagggcggttttttcctgtttggtcactgatgcctccgtgtaagggggatttctgttcatgggggtaatgataccgatgaaacgagagaggatgctcacgatacgggttactgatgatgaacatgcccggttactggaacgttgtgagggtaaacaactggcggtatggatgcggcgggaccagagaaaaatcactcagggtcaatgccagcgcttcgttaatacagatgtaggtgttccacagggtagccagcagcatcctgcgatgcagatccggaacataatggtgcagggcgctgacttccgcgtttccagactttacgaaacacggaaaccgaagaccattcatgttgttgctcaggtcgcagacgttttgcagcagcagtcgcttcacgttcgctcgcgtatcggtgattcattctgctaaccagtaaggcaaccccgccagcctagccgggtcctcaacgacaggagcacgatcatgcgcacccgtggggccgccatgccggcgataatggcctgcttctcgccgaaacgtttggtggcgggaccagtgacgaaggcttgagcgagggcgtgcaagattccgaataccgcaagcgacaggccgatcatcgtcgcgctccagcgaaagcggtcctcgccgaaaatgacccagagcgctgccggcacctgtcctacgagttgcatgataaagaagacagtcataagtgcggcgacgatagtcatgccccgcgcccaccggaaggagctgactgggttgaaggctctcaagggcatcggtcgagatcccggtgcctaatgagtgagctaacttacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgccagggtggtttttcttttcaccagtgagacgggcaacagctgattgcccttcaccgcctggccctgagagagttgcagcaagcggtccacgctggtttgccccagcaggcgaaaatcctgtttgatggtggttaacggcgggatataacatgagctgtcttcggtatcgtcgtatcccactaccgagatatccgcaccaacgcgcagcccggactcggtaatggcgcgcattgcgcccagcgccatctgatcgttggcaaccagcatcgcagtgggaacgatgccctcattcagcatttgcatggtttgttgaaaaccggacatggcactccagtcgccttcccgttccgctatcggctgaatttgattgcgagtgagatatttatgccagccagccagacgcagacgcgccgagacagaacttaatgggcccgctaacagcgcgatttgctggtgacccaatgcgaccagatgctccacgcccagtcgcgtaccgtcttcatgggagaaaataatactgttgatgggtgtctggtcagagacatcaagaaataacgccggaacattagtgcaggcagcttccacagcaatggcatcctggtcatccagcggatagttaatgatcagcccactgacgcgttgcgcgagaagattgtgcaccgccgctttacaggcttcgacgccgcttcgttctaccatcgacaccaccacgctggcacccagttgatcggcgcgagatttaatcgccgcgacaatttgcgacggcgcgtgcagggccagactggaggtggcaacgccaatcagcaacgactgtttgcccgccagttgttgtgccacgcggttgggaatgtaattcagctccgccatcgccgcttccactttttcccgcgttttcgcagaaacgtggctggcctggttcaccacgcgggaaacggtctgataagagacaccggcatactctgcgacatcgtataacgttactggtttcacattcaccaccctgaattgactctcttccgggcgctatcatgccataccgcgaaaggttttgcgccattcgatggtgtccgggatctcgacgctctcccttatgcgactcctgcattaggaagcagcccagtagtaggttgaggccgttgagcaccgccgccgcaaggaatggtgcatgcaaggagatggcgcccaacagtcccccggccacggggcctgccaccatacccacgccgaaacaagcgctcatgagcccgaagtggcgagcccgatcttccccatcggtgatgtcggcgatataggcgccagcaaccgcacctgtggcgccggtgatgccggccacgatgcgtccggcgtagaggatcg 25agatctcgatcccgcgaaattaatacgactcactataggggaattgtgagcggataacaattcccctctagaaataattttgtttaactttaagaEndo-β- aggagatataCATATGGTACCAAACAAAAGCGTAGCACCATTATTACTTGCTGCATCTATglucanase CTTATATGGTGGTGCTGTTGCTCAACAGACTGTTTGGGGTCAGTGTGGTGGTATTGGinsertion cassetteTTGGTCTGGTCCTACCAATTGTGCTCCTGGCTCAGCATGTAGTACCTTAAATCCTTA (pET-21a-BD05)CTATGCTCAATGTATTCCAGGTGCAACAACTATAACAACATCAACTCGCCCTCCTTCAGGTCCAACTACAACAACTCGTGCTACTAGCACTTCTAGCAGCACACCTCCTACATCTTCTGGAGTACGTTTCGCTGGTGTTAATATTGCAGGTTTCGATTTTGGTTGTACTACCGATGGTACATGTGTTACCAGTAAAGTTTATCCCCCTTTAAAAAATTTTACTGGCTCAAACAATTATCCAGATGGCATTGGTCAAATGCAACACTTTGTAAATGAAGATGGTATGACTATTTTCCGTTTACCAGTGGGCTGGCAATACTTAGTTAACAACAATTTAGGTGGTAACTTAGATAGTACATCAATTAGTAAATATGATCAATTAGTACAAGGTTGCTTATCTTTAGGTGCCTATTGTATTGTTGATATTCATAATTATGCCCGTTGGAACGGTGGTATTATTGGTCAAGGTGGTCCAACTAATGCTCAATTTACATCATTATGGAGCCAATTAGCTTCAAAATATGCTAGTCAATCACGTGTTTGGTTCGGTATTATGAATGAACCTCACGATGTGAACATAAATACTTGGGCTGCAACTGTGCAAGAAGTAGTAACTGCTATTCGTAATGCTGGTGCAACATCACAATTCATTAGTTTACCAGGCAACGATTGGCAATCTGCCGGCGCTTTTATTTCTGACGGTAGCGCAGCTGCTCTTAGTCAAGTGACTAACCCAGACGGTAGTACCACTAACTTAATATTCGATGTACATAAATATCTTGATTCTGATAATAGCGGAACACACGCCGAATGTACCACAAATAATATTGATGGTGCTTTTAGTCCTTTAGCAACTTGGTTACGTCAAAATAATCGCCAAGCCATTTTAACTGAAACAGGTGGTGGAAACGTGCAGAGTTGTATCCAAGACATGTGTCAACAAATTCAGTACTTAAATCAAAACTCTGACGTGTACTTAGGTTATGTAGGTTGGGGTGCTGGTTCTTTTGATTCAACTTATGTATTAACCGAAACCCCTACTTCTTCTGGAAACTCATGGACAGACACTTCATTAGTAAGTAGTTGTTTAGCTCGCAAGGGTACCGGTGAAAACTTATACTTTCAAGGCTCAGGTGGCGGTGGAAGTGATTACAAAGATGATGATGATAAAGGAACCGGTTAATCTAGACTCGAGcaccaccaccaccaccactgagatccggctgctaacaaagcccgaaaggaagctgagttggctgctgccaccgctgagcaataactagcataaccccttggggcctctaaacgggtcttgaggggttttttgctgaaaggaggaactatatccggattggcgaatgggacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgtttacaatttcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgcagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcctgatgcggtattttctccttacgcatctgtgcggtatttcacaccgcatatatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagtatacactccgctatcgctacgtgactgggtcatggctgcgccccgacacccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttacagacaagctgtgaccgtctccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgaggcagctgcggtaaagctcatcagcgtggtcgtgaagcgattcacagatgtctgcctgttcatccgcgtccagctcgttgagtttctccagaagcgttaatgtctggcttctgataaagcgggccatgttaagggcggttttttcctgtttggtcactgatgcctccgtgtaagggggatttctgttcatgggggtaatgataccgatgaaacgagagaggatgctcacgatacgggttactgatgatgaacatgcccggttactggaacgttgtgagggtaaacaactggcggtatggatgcggcgggaccagagaaaaatcactcagggtcaatgccagcgcttcgttaatacagatgtaggtgttccacagggtagccagcagcatcctgcgatgcagatccggaacataatggtgcagggcgctgacttccgcgtttccagactttacgaaacacggaaaccgaagaccattcatgttgttgctcaggtcgcagacgttttgcagcagcagtcgcttcacgttcgctcgcgtatcggtgattcattctgctaaccagtaaggcaaccccgccagcctagccgggtcctcaacgacaggagcacgatcatgcgcacccgtggggccgccatgccggcgataatggcctgcttctcgccgaaacgtttggtggcgggaccagtgacgaaggcttgagcgagggcgtgcaagattccgaataccgcaagcgacaggccgatcatcgtcgcgctccagcgaaagcggtcctcgccgaaaatgacccagagcgctgccggcacctgtcctacgagttgcatgataaagaagacagtcataagtgcggcgacgatagtcatgccccgcgcccaccggaaggagctgactgggttgaaggctctcaagggcatcggtcgagatcccggtgcctaatgagtgagctaacttacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgccagggtggtttttcttttcaccagtgagacgggcaacagctgattgcccttcaccgcctggccctgagagagttgcagcaagcggtccacgctggtttgccccagcaggcgaaaatcctgtttgatggtggttaacggcgggatataacatgagctgtcttcggtatcgtcgtatcccactaccgagatatccgcaccaacgcgcagcccggactcggtaatggcgcgcattgcgcccagcgccatctgatcgttggcaaccagcatcgcagtgggaacgatgccctcattcagcatttgcatggtttgttgaaaaccggacatggcactccagtcgccttcccgttccgctatcggctgaatttgattgcgagtgagatatttatgccagccagccagacgcagacgcgccgagacagaacttaatgggcccgctaacagcgcgatttgctggtgacccaatgcgaccagatgctccacgcccagtcgcgtaccgtcttcatgggagaaaataatactgttgatgggtgtctggtcagagacatcaagaaataacgccggaacattagtgcaggcagcttccacagcaatggcatcctggtcatccagcggatagttaatgatcagcccactgacgcgttgcgcgagaagattgtgcaccgccgctttacaggcttcgacgccgcttcgttctaccatcgacaccaccacgctggcacccagttgatcggcgcgagatttaatcgccgcgacaatttgcgacggcgcgtgcagggccagactggaggtggcaacgccaatcagcaacgactgtttgcccgccagttgttgtgccacgcggttgggaatgtaattcagctccgccatcgccgcttccactttttcccgcgttttcgcagaaacgtggctggcctggttcaccacgcgggaaacggtctgataagagacaccggcatactctgcgacatcgtataacgttactggtttcacattcaccaccctgaattgactctcttccgggcgctatcatgccataccgcgaaaggttttgcgccattcgatggtgtccgggatctcgacgctctcccttatgcgactcctgcattaggaagcagcccagtagtaggttgaggccgttgagcaccgccgccgcaaggaatggtgcatgcaaggagatggcgcccaacagtcccccggccacggggcctgccaccatacccacgccgaaacaagcgctcatgagcccgaagtggcgagcccgatcttccccatcggtgatgtcggcgatataggcgccagcaaccgcacctgtggcgccggtgatgccggccacgatgcgtccggcgtagaggatcg 26agatctcgatcccgcgaaattaatacgactcactataggggaattgtgagcggataacaattcccctctagaaataattttgtttaactttaagaβ-glucosidaseaggagatataCATATGGTACCATTACCAAAGGATTTCCAATGGGGTTTCGCTACCGCAGC insertionTTATCAAATTGAAGGTGCAGTTGATCAAGATGGACGTGGACCTTCTATTTGGGACA cassetteCATTCTGTGCACAACCAGGTAAAATTGCTGATGGTTCATCAGGTGTAACAGCATGT (pET-21a-GACTCATATAATCGTACAGCTGAAGACATTGCACTTTTAAAATCTTTAGGTGCTAAA BD09)TCATATCGTTTCTCTATCTCATGGTCAAGAATTATTCCTGAAGGTGGCCGTGGTGACGCAGTAAATCAAGCTGGTATTGATCACTATGTTAAATTTGTAGATGACTTATTAGACGCAGGTATTACACCTTTTATCACTTTATTTCACTGGGATTTACCTGAAGGTTTACACCAACGTTATGGTGGTCTTTTAAACCGTACAGAATTTCCTTTAGATTTCGAAAACTATGCAAGAGTTATGTTTCGTGCACTTCCCAAAGTAAGAAACTGGATTACTTTTAATGAACCTTTATGTTCTGCTATTCCTGGTTATGGTTCAGGCACCTTTGCCCCAGGCAGACAAAGTACAAGTGAGCCCTGGACAGTGGGCCATAACATTTTAGTAGCTCACGGTAGAGCTGTAAAAGCATATAGAGATGATTTCAAACCTGCTTCAGGTGATGGTCAAATAGGTATTGTGTTAAATGGTGACTTCACATATCCCTGGGATGCCGCTGATCCTGCAGATAAAGAAGCCGCTGAACGTCGCTTAGAATTTTTCACTGCTTGGTTTGCTGACCCCATCTATCTTGGTGATTATCCTGCTTCAATGCGTAAACAATTAGGTGATCGTTTACCTACTTTTACACCAGAAGAACGTGCTTTAGTTCATGGTAGTAATGACTTTTATGGTATGAACCACTATACTTCAAACTATATTCGTCACCGTAGCTCACCCGCAAGTGCTGATGACACAGTAGGTAATGTAGATGTTTTATTTACTAATAAACAAGGTAATTGTATCGGTCCTGAAACACAGAGCCCCTGGCTTCGTCCTTGTGCAGCTGGTTTCCGTGACTTCCTTGTATGGATAAGCAAACGTTATGGTTATCCACCAATTTATGTTACAGAAAACGGAACATCAATAAAAGGTGAAAGTGACTTACCAAAGGAAAAGATTCTTGAAGATGATTTTCGTGTTAAGTATTATAACGAATACATTAGAGCTATGGTTACAGCCGTTGAATTAGATGGTGTAAATGTAAAAGGTTATTTCGCATGGTCTTTAATGGATAACTTTGAATGGGCTGATGGTTACGTTACACGTTTTGGTGTAACCTACGTTGATTACGAAAACGGCCAAAAACGTTTCCCTAAAAAGAGTGCTAAAAGTTTAAAACCTTTATTTGATGAATTAATAGCTGCTGCAGGTACCGGTGAAAACTTATACTTTCAAGGCTCAGGTGGCGGTGGAAGTGATTACAAAGATGATGATGATAAAGGAACCGGTTAATCTAGACTCGAGcaccaccaccaccaccactgagatccggctgctaacaaagcccgaaaggaagctgagttggctgctgccaccgctgagcaataactagcataaccccttggggcctctaaacgggtcttgaggggttttttgctgaaaggaggaactatatccggattggcgaatgggacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgtttacaatttcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgcagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcctgatgcggtattttctccttacgcatctgtgcggtatttcacaccgcatatatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagtatacactccgctatcgctacgtgactgggtcatggctgcgccccgacacccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttacagacaagctgtgaccgtctccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgaggcagctgcggtaaagctcatcagcgtggtcgtgaagcgattcacagatgtctgcctgttcatccgcgtccagctcgttgagtttctccagaagcgttaatgtctggcttctgataaagcgggccatgttaagggcggttttttcctgtttggtcactgatgcctccgtgtaagggggatttctgttcatgggggtaatgataccgatgaaacgagagaggatgctcacgatacgggttactgatgatgaacatgcccggttactggaacgttgtgagggtaaacaactggcggtatggatgcggcgggaccagagaaaaatcactcagggtcaatgccagcgcttcgttaatacagatgtaggtgttccacagggtagccagcagcatcctgcgatgcagatccggaacataatggtgcagggcgctgacttccgcgtttccagactttacgaaacacggaaaccgaagaccattcatgttgttgctcaggtcgcagacgttttgcagcagcagtcgcttcacgttcgctcgcgtatcggtgattcattctgctaaccagtaaggcaaccccgccagcctagccgggtcctcaacgacaggagcacgatcatgcgcacccgtggggccgccatgccggcgataatggcctgcttctcgccgaaacgtttggtggcgggaccagtgacgaaggcttgagcgagggcgtgcaagattccgaataccgcaagcgacaggccgatcatcgtcgcgctccagcgaaagcggtcctcgccgaaaatgacccagagcgctgccggcacctgtcctacgagttgcatgataaagaagacagtcataagtgcggcgacgatagtcatgccccgcgcccaccggaaggagctgactgggttgaaggctctcaagggcatcggtcgagatcccggtgcctaatgagtgagctaacttacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgccagggtggtttttcttttcaccagtgagacgggcaacagctgattgcccttcaccgcctggccctgagagagttgcagcaagcggtccacgctggtttgccccagcaggcgaaaatcctgtttgatggtggttaacggcgggatataacatgagctgtcttcggtatcgtcgtatcccactaccgagatatccgcaccaacgcgcagcccggactcggtaatggcgcgcattgcgcccagcgccatctgatcgttggcaaccagcatcgcagtgggaacgatgccctcattcagcatttgcatggtttgttgaaaaccggacatggcactccagtcgccttcccgttccgctatcggctgaatttgattgcgagtgagatatttatgccagccagccagacgcagacgcgccgagacagaacttaatgggcccgctaacagcgcgatttgctggtgacccaatgcgaccagatgctccacgcccagtcgcgtaccgtcttcatgggagaaaataatactgttgatgggtgtctggtcagagacatcaagaaataacgccggaacattagtgcaggcagcttccacagcaatggcatcctggtcatccagcggatagttaatgatcagcccactgacgcgttgcgcgagaagattgtgcaccgccgctttacaggcttcgacgccgcttcgttctaccatcgacaccaccacgctggcacccagttgatcggcgcgagatttaatcgccgcgacaatttgcgacggcgcgtgcagggccagactggaggtggcaacgccaatcagcaacgactgtttgcccgccagttgttgtgccacgcggttgggaatgtaattcagctccgccatcgccgcttccactttttcccgcgttttcgcagaaacgtggctggcctggttcaccacgcgggaaacggtctgataagagacaccggcatactctgcgacatcgtataacgttactggtttcacattcaccaccctgaattgactctcttccgggcgctatcatgccataccgcgaaaggttttgcgccattcgatggtgtccgggatctcgacgctctcccttatgcgactcctgcattaggaagcagcccagtagtaggttgaggccgttgagcaccgccgccgcaaggaatggtgcatgcaaggagatggcgcccaacagtcccccggccacggggcctgccaccatacccacgccgaaacaagcgctcatgagcccgaagtggcgagcccgatcttccccatcggtgatgtcggcgatataggcgccagcaaccgcacctgtggcgccggtgatgccggccacgatgcgtccggcgtagaggatcg27agatctcgatcccgcgaaattaatacgactcactataggggaattgtgagcggataacaattcccctctagaaataattttgtttaactttaagaEndo- aggagatataCATATGGTACCAGTATCTTTCACAAGTCTTTTAGCAGCATCTCCACCTTCAxylanase CGTGCAAGTTGCCGTCCAGCTGCTGAAGTGGAATCAGTTGCAGTAGAAAAACGTCAinsertion AACAATTCAACCAGGTACAGGTTACAATAACGGTTACTTTTATTCTTACTGGAATGAcassette TGGACACGGTGGTGTTACATATACTAATGGACCTGGTGGTCAATTTAGTGTAAATTG(pET-21a- GAGTAACTCAGGCAATTTTGTTGGAGGAAAAGGTTGGCAACCTGGTACAAAGAATA BD11)AGGTAATCAATTTCTCTGGTAGTTACAACCCTAATGGTAATTCTTATTTAAGTGTATACGGTTGGAGCCGTAACCCATTAATTGAATATTATATTGTAGAGAACTTTGGTACATACAACCCTTCAACAGGTGCTACTAAATTAGGTGAAGTTACTTCAGATGGATCAGTTTATGATATTTATCGTACTCAACGCGTAAATCAACCATCTATAATTGGAACTGCCACTTTCTACCAATACTGGAGTGTAAGACGTAATCATCGTTCAAGTGGTAGTGTTAATACAGCAAACCACTTTAATGCATGGGCTCAACAAGGTTTAACATTAGGTACAATGGACTATCAAATTGTAGCTGTTGAAGGTTATTTTTCATCAGGTAGTGCTTCTATCACTGTTAGCGGTACCGGTGAAAACTTATACTTTCAAGGCTCAGGTGGCGGTGGAAGTGATTACAAAGATGATGATGATAAAGGAACCGGTTAATCTAGACTCGAGcaccaccaccaccaccactgagatccggctgctaacaaagcccgaaaggaagctgagttggctgctgccaccgctgagcaataactagcataaccccttggggcctctaaacgggtcttgaggggttttttgctgaaaggaggaactatatccggattggcgaatgggacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgtttacaatttcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgcagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcctgatgcggtattttctccttacgcatctgtgcggtatttcacaccgcatatatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagtatacactccgctatcgctacgtgactgggtcatggctgcgccccgacacccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttacagacaagctgtgaccgtctccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgaggcagctgcggtaaagctcatcagcgtggtcgtgaagcgattcacagatgtctgcctgttcatccgcgtccagctcgttgagtttctccagaagcgttaatgtctggcttctgataaagcgggccatgttaagggcggttttttcctgtttggtcactgatgcctccgtgtaagggggatttctgttcatgggggtaatgataccgatgaaacgagagaggatgctcacgatacgggttactgatgatgaacatgcccggttactggaacgttgtgagggtaaacaactggcggtatggatgcggcgggaccagagaaaaatcactcagggtcaatgccagcgcttcgttaatacagatgtaggtgttccacagggtagccagcagcatcctgcgatgcagatccggaacataatggtgcagggcgctgacttccgcgtttccagactttacgaaacacggaaaccgaagaccattcatgttgttgctcaggtcgcagacgttttgcagcagcagtcgcttcacgttcgctcgcgtatcggtgattcattctgctaaccagtaaggcaaccccgccagcctagccgggtcctcaacgacaggagcacgatcatgcgcacccgtggggccgccatgccggcgataatggcctgcttctcgccgaaacgtttggtggcgggaccagtgacgaaggcttgagcgagggcgtgcaagattccgaataccgcaagcgacaggccgatcatcgtcgcgctccagcgaaagcggtcctcgccgaaaatgacccagagcgctgccggcacctgtcctacgagttgcatgataaagaagacagtcataagtgcggcgacgatagtcatgccccgcgcccaccggaaggagctgactgggttgaaggctctcaagggcatcggtcgagatcccggtgcctaatgagtgagctaacttacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgccagggtggtttttcttttcaccagtgagacgggcaacagctgattgcccttcaccgcctggccctgagagagttgcagcaagcggtccacgctggtttgccccagcaggcgaaaatcctgtttgatggtggttaacggcgggatataacatgagctgtcttcggtatcgtcgtatcccactaccgagatatccgcaccaacgcgcagcccggactcggtaatggcgcgcattgcgcccagcgccatctgatcgttggcaaccagcatcgcagtgggaacgatgccctcattcagcatttgcatggtttgttgaaaaccggacatggcactccagtcgccttcccgttccgctatcggctgaatttgattgcgagtgagatatttatgccagccagccagacgcagacgcgccgagacagaacttaatgggcccgctaacagcgcgatttgctggtgacccaatgcgaccagatgctccacgcccagtcgcgtaccgtcttcatgggagaaaataatactgttgatgggtgtctggtcagagacatcaagaaataacgccggaacattagtgcaggcagcttccacagcaatggcatcctggtcatccagcggatagttaatgatcagcccactgacgcgttgcgcgagaagattgtgcaccgccgctttacaggcttcgacgccgcttcgttctaccatcgacaccaccacgctggcacccagttgatcggcgcgagatttaatcgccgcgacaatttgcgacggcgcgtgcagggccagactggaggtggcaacgccaatcagcaacgactgtttgcccgccagttgttgtgccacgcggttgggaatgtaattcagctccgccatcgccgcttccactttttcccgcgttttcgcagaaacgtggctggcctggttcaccacgcgggaaacggtctgataagagacaccggcatactctgcgacatcgtataacgttactggtttcacattcaccaccctgaattgactctcttccgggcgctatcatgccataccgcgaaaggttttgcgccattcgatggtgtccgggatctcgacgctctcccttatgcgactcctgcattaggaagcagcccagtagtaggttgaggccgttgagcaccgccgccgcaaggaatggtgcatgcaaggagatggcgcccaacagtcccccggccacggggcctgccaccatacccacgccgaaacaagcgctcatgagcccgaagtggcgagcccgatcttccccatcggtgatgtcggcgatataggcgccagcaaccgcacctgtggcgccggtgatgccggccacgatgcgtccggcgtagaggatcg28 GTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGCCCCGACACCC Endo-β-GCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACA glucanaseGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCAC insertionCGAAACGCGCGAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTC cassetteATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGG (pSE-3HB-AACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACA K-rbcL:ATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAAC BD05)ATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCaagctcgcggccgcagtactCTGCAGATTTTATGCAAAATTAAAGTCTTGTGACAACAGCTTTCTCCTTAAGTGCAAATATCGCCCATTCTTTCCTCTTTTCGTATATAAATGCTGTAATAGTAGGATGTCGTACCCGTAAAGGTACGACATTGAATATTAATATACTCCTAAGTTTACTTTCCCAATATTTATATTAGGACGTCCCCTTCGGGTAAATAAATTTTAGTGGCAGTGGTACCGCCACTCCCTATTTTAATACTGCGAAGGAGGCAGTTGGCAGGCAACTCGTCGTTCGCAGTATATAAATATCCACTAATATTTATATTCCCGTAAGGGGACGTCCCGAAGGGGAAGGGGAAAGAAGCAGTCGCCTCCTTGCGAAAAGGTTTACTTGCCCGACCAGTGAAAAGCATGCTGTAAGATATAAATCTACCCTGAAAGGGATGCATTTCACCATAATACTATACAAATGGTGTTACCCTTTGAGGATCATAACGGTGCTACTGGAATATATGGTCTCTTCATGGATAGACGATAGCCATTTATTTACCCATTAAGGGGACATTAGTGGCCTGTCACTGCTCCTTACGAGACGCCAGTGGACGTTCGTCCTAGAAAATTTATGCGCTGCCTAGAAGCCCCAAAAGGGAAGTTTACTGACTCGTTAGAGCGTGCGCTAACAGGTTTAAATACTTCAATATGTATATTAGGACGCCGGTGGCAGTGGTACCGCCACTGCCACCGTCGGAGGACGTCCCTTACGGTATATTATATACTAGGATTTTAATACTCCGAAGGAGGCAGTGGCGGTACCACTGCCACTAATATTTATATTCCCGTAAGGGACGTCCTCCTTCGGAGTATGTAAACATTCTAAGTTTACTTGCCCAATATTTATATTAGGCAGTTGGCAGGCAACTGCTAGCTCTCCTCCTTCGGAGTATGTAAACATCGCAGTATATAAATATCCACTAATATTTATATTCCCGTAAGGGGACGTCCCGAAGGGGAAGGGGAAGGACGTCAGTGGCAGTTGCCTGCCAACTGCCTAGGCAAGTAAACTTAGGAGTATATAAATATAGGCAGTCGCGGTACCACTGCCACTGACGTCCTGCCAACTGCCTAGGCAAGTAAACTTAAGTGGCACTAAAATGCATTTGCCCGAAGGGGAAGGAGGACGCCAGTGGCAGTGGTACCGCCACTGCCTCCTTCGGAGTATTAAAATCCTAGTATGTAAATCTGCTAGCGCAGGAAATAAATTTTATTCTATTTATATACTCCGTTAGGAGGTAAGTAAACCCCTTCCCCTTCGGGACGTCAGTGCAGTTGCCTGCCAACTGCCTAATATAAATATTAGACCACTAAAGTTTGGCAACTGCCAACTGTTGTCCTTCGGAGGAAAAAAAATGGTTAACTCGCAAGCAGTTAACATAACTAAAGTTTGTTACTTTACCGAAGACGTTTACCCTTTCTCGGTTAAGGAGACGGAGACAGTTGCACTGTGACTGCCTAGTATAGCAATTTTGTTTTTGTTTATATGCTCGACAAAATGACTTTCATAAAAATATAAAGTAGTTAGCTAGTTATTTTATATCACTATAACTAGGGTTCTCAGAGGCACCGAAGTCACTTGTAAAAATAGTACTTTTTAACTTGTTTAATCTTCGTGTTCTTCAAAAGGATCACGTAATTTTTTTGAAGGTGGACCAAAACTAACATAAACTGAATAGCCAGTTACACTTAACAGAAGAAACCATAAAAAAAAGGTAAAGAAAAAAGCTGGACTTTCCATAGCTCATTTAATAATAAAATTATTCTCTTTTCAACATATCTCTTAGATAGTTCAAAAGACTTGACGACTGTGTCCCACATTTTTAAACAAAATTAATCTACTCAAAATTTTGCCCTGAGAAAGAATAACTTACTTCGTTTTTGCAGTAGCCATTCATGTCACTTTGAAACTGTCCTTACAAAGTTAAACATTAATTAAAAATTATTTAATTTTTATATAACAAATATTATATTAAATAAAAAATGAACAAAGAACTTCTAAGATCGTCTTTAGTGAGTAATTAAAGAGTTTTACTTACCAGACAAGGCAGTTTTTTCATTCTTTTAAAGCAGGCAGTTCTGAAGGGGAAAAGGGACTGCCTACTGCGGTCCTAGGTAAATACATTTTTATGCAATTTATTTCTTGTGCTAGTAGGTTTCTATACTCACAAGAAGCAACCCCTTGACGAGAGAACGTTATCCTCAGAGTATTTATAATCCTGAGAGGGAATGCACTGAAGAATATTTTCCTTATTTTTTACAGAAAGTAAATAAAATAGCGCTAATAACGCTTAATTCATTTAATCAATTATGGCAACAGGAACTTCTAAAGCTAAACCATCAAAAGTAAATTCAGACTTCCAAGAACCTGGTTTAGTTACACCATTAGGTACTTTATTACGTCCACTTAACTCAGAAGCAGGTAAAGTATTACCAGGCTGGGGTACAACTGTTTTAATGGCTGTATTTATCCTTTTATTTGCAGCATTCTTATTAATCATTTTAGAAATTTACAACAGTTCTTTAATTTTAGATGACGTTTCTATGAGTTGGGAAACTTTAGCTAAAGTTTCTTAATTTTATTTAACACAAACATAAAATATAAAACTGTTTGTTAAGGCTAGCTGCTAAGTCTTCTTTTCGCTAAGGTAAACTAAGCAACTCAACCATATTTATATTCGGCAGTGGCACCGCCAACTGCCACTGGCCTTCCGTTAAGATAAACGCGTggatctcacgtgACTAGTcacctagtgtcgagtggtaccgccactgcctagtatataaatatcggcagttggcaggatatttatatactccgaaggaacttgttagccgataggcgaggcaactgccactaaaatttatttgcctcctaacggagcattaaaatccctaagtttacttgcccgtaaggggaaggggacgtccactaatatttatattaggcagttggcaggcaacaataaatacatttgtcccgtaaggggacgtcctgccaactgcctatggtagctattaagtatatatatatgaaaagtgtgtataaactaaactaaaataaaccaggtatggttaaccagatttattttagtttaaaaaaaaattagttgtttgagctagagttagttgaagctaagtctagaTTAACCGGTTCCTTTATCATCATCATCTTTGTAATCACTTCCACCGCCACCTGAGCCTTGAAAGTATAAGTTTTCACCGGTACCCTTGCGAGCTAAACAACTACTTACTAATGAAGTGTCTGTCCATGAGTTTCCAGAAGAAGTAGGGGTTTCGGTTAATACATAAGTTGAATCAAAAGAACCAGCACCCCAACCTACATAACCTAAGTACACGTCAGAGTTTTGATTTAAGTACTGAATTTGTTGACACATGTCTTGGATACAACTCTGCACGTTTCCACCACCTGTTTCAGTTAAAATGGCTTGGCGATTATTTTGACGTAACCAAGTTGCTAAAGGACTAAAAGCACCATCAATATTATTTGTGGTACATTCGGCGTGTGTTCCGCTATTATCAGAATCAAGATATTTATGTACATCGAATATTAAGTTAGTGGTACTACCGTCTGGGTTAGTCACTTGACTAAGAGCAGCTGCGCTACCGTCAGAAATAAAAGCGCCGGCAGATTGCCAATCGTTGCCTGGTAAACTAATGAATTGTGATGTTGCACCAGCATTACGAATAGCAGTTACTACTTCTTGCACAGTTGCAGCCCAAGTATTTATGTTCACATCGTGAGGTTCATTCATAATACCGAACCAAACACGTGATTGACTAGCATATTTTGAAGCTAATTGGCTCCATAATGATGTAAATTGAGCATTAGTTGGACCACCTTGACCAATAATACCACCGTTCCAACGGGCATAATTATGAATATCAACAATACAATAGGCACCTAAAGATAAGCAACCTTGTACTAATTGATCATATTTACTAATTGATGTACTATCTAAGTTACCACCTAAATTGTTGTTAACTAAGTATTGCCAGCCCACTGGTAAACGGAAAATAGTCATACCATCTTCATTTACAAAGTGTTGCATTTGACCAATGCCATCTGGATAATTGTTTGAGCCAGTAAAATTTTTTAAAGGGGGATAAACTTTACTGGTAACACATGTACCATCGGTAGTACAACCAAAATCGAAACCTGCAATATTAACACCAGCGAAACGTACTCCAGAAGATGTAGGAGGTGTGCTGCTAGAAGTGCTAGTAGCACGAGTTGTTGTAGTTGGACCTGAAGGAGGGCGAGTTTGATGTTGTTATAGTTGTTGCACCTGGAATACATTGAGCATAGTAAGGATTTAAGGTACTACATGCTGAGCCAGGAGCACAATTGGTAGGACCAGACCAACCAATACCACCACACTGACCCCAAACAGTCTGTTGAGCAACAGCACCACCATATAAGATAGATGCAGCAAGTAATAATGGTGCTACGCTTTTGTTTGGTACCATatgcactttgcattacctccgtacaaattattttgatttctataaagttttgcttaaataaaaatttttaatttttaacgtccaccatataaataataaatatggtgaaacctttaacaacaaaaatcctcttgtaccatattaatccaaaagaattaaggacaaaagcttatctccaacatttttaaaacacagagtaaaaataatgttgtttttaagaatagaattttataacttgtattttaaatatgatctaatttatttgtgctaaaaattgcagttggaaagtaattttaaaaataatttagatcatatttattaaataaagttgatttaaaacaacttaatcgtttttaattgttaattaaaaacataattttaaatctttttatatttaaattaccttatatactactaggtgACTATGgatatctacgtaatcgatgaattcgatcccatttttataactggatctcaaaatacctataaacccattgttcttctcttttagctctaagaacaatcaatttataaatatatttattattatgctataatataaatactatataaatacatttacctttttataaatacatttaccttttttttaatttgcatgattttaatgcttatgctatcttttttatttagtccataaaacctttaaaggaccttttcttatgggatatttatattttcctaacaaagcaatcggcgtcataaactttagttgcttacgacgcctgtggacgtcccccccttccccttacgggcaagtaaacttagggattttaatgcaataaataaatttgtcctcttcgggcaaatgaattttagtatttaaatatgacaagggtgaaccattacttttgttaacaagtgatcttaccactcactatttttgttgaattttaaacttatttaaaattctcgaaagattttaaaaataaacttttttaatcttttatttattttttcttttttCGTATGGAATTGCCCAATATTATTCAACAATTTATCGGAAACAGCGTTTTAGAGCCAAATAAAATTGGTCAGTCGCCATCGGATGTTTATTCTTTTAATCGAAATAATGAAACTTTTTTTCTTAAGCGATCTAGCACTTTATATACAGAGACCACATACAGTGTCTCTCGTGAAGCGAAAATGTTGAGTTGGCTCTCTGAGAAATTAAAGGTGCCTGAACTCATCATGACTTTTCAGGATGAGCAGTTTGAATTTATGATCACTAAAGCGATCAATGCAAAACCAATTTCAGCGCTTTTTTTAACAGACCAAGAATTGCTTGCTATCTATAAGGAGGCACTCAATCTGTTAAATTCAATTGCTATTATTGATTGTCCATTTATTTCAAACATTGATCATCGGTTAAAAGAGTCAAAATTTTTTATTGATAACCAACTCCTTGACGATATAGATCAAGATGATTTTGACACTGAATTATGGGGAGACCATAAAACTTACCTAAGTCTATGGAATGAGTTAACCGAGACTCGTGTTGAAGAAAGATTGGTTTTTTCTCATGGCGATATCACGGATAGTAATATTTTTATAGATAAATTCAATGAAATTTATTTTTTAGACCTTGGTCGTGCTGGGTTAGCAGATGAATTTGTAGATATATCCTTTGTTGAACGTTGCCTAAGAGAGGATGCATCGGAGGAAACTGCGAAAATATTTTTAAAGCATTTAAAAAATGATAGACCTGACAAAAGGAATTATTTTTTAAAACTTGATGAATTGAATTGAttccaagcattatctaaaatactctgcaggcacgctagcttgtactcaagctcgtaacgaaggtcgtgaccttgctcgtgaaggtggcgacgtaattcgttcagcttgtaaatggtctccagaacttgctgctgcatgtgaagtttggaaagaaattaaattcgatttgatactattgacaaactttaatttttatttttcatgatgtttatgaatagcataaacatcgtttttatttttatggtgtttaggttaaatacctaaacatcattttacatttttaaaattaagttctaaagttatcttttgtttaaatttgcctgtctttatattacgatgtgccagaaaaataaaatcttagctttttattatagaatttatctttatgtattatattttataagttataataaaagaaatagtaacatactaaagcggatgtagcgcgtttatcttaacggaaggaattcggcgcctacgtacccgggtcgcgaggatccACGCGTTAATAGCTCACTTTTCTTTAAATTTAATTTTTAATTTAAAGGTGTAAGCAAATTGCCTGACGAGAGATCCACTTAAAGGATGACAGTGGCGGGCTACTGCCTACTTCCCTCCGGGATAAAATTTATTTGAAAAACGTTAGTTACTTCCTAACGGAGCATTGACATCCCCATATTTATATTAGGACGTCCCCTTCGGGTAAATAAATTTTAGTGGACGTCCCCTTCGGGCAAATAAATTTTAGTGGACAATAAATAAATTTGTTGCCTGCCAACTGCCTAGGCAAGTAAACTTGGGAGTATTAAAATAGGACGTCAGTGGCAGTTGCCTGCCAACTGCCTATATTTATATACTGCGAAGCAGGCAGTGGCGGTACCACTGCCACTGGCGTCCTAATATAAATATTGGGCAACTAAAGTTTATAGCAGTATTAACATCCTATATTTATATACTCCGAAGGAACTTGTTAGCCGATAGGCGAGGCAACAAATTTATTTATTGTCCCGTAAAAGGATGCCTCCAGCATCGAAGGGGAAGGGGACGTCCTAGGCCATAAAACTAAAGGGAAATCCATAGTAACTGATGTTATAAATTTATAGACTCCAAAAAACAGCTGCGTTATAAATAACTTCTGTTAAATATGGCCAAGGGGACAGGGGCACTTTCAACTAAGTGTACATTAAAAATTGACAATTCAATTTTTTTTAATTATAATATATATTTAGTAAAATATAACAAAAAGCCCCCATCGTCTAGgtagaattccagctggcggccgccctatg 29GTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGCCCCGACACCC Exo-β-GCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACA glucanaseGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCAC insertionCGAAACGCGCGAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTC cassetteATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGG (pSE-3HB-AACCCCTATTTGTTTATTTTTCTAAATACATTTCAAATATGTATCCGCTCATGAGACA K-tD2:ATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAAC BD01)ATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCaagctcgcggccgcagtactCTGCAGATTTTATGCAAAATTAAAGTCTTGTGACAACAGCTTTCTCCTTAAGTGCAAATATCGCCCATTCTTTCCTCTTTTCGTATATAAATGCTGTAATAGTAGGATGTCGTACCCGTAAAGGTACGACATTGAATATTAATATACTCCTAAGTTTACTTTCCCAATATTTATATTAGGACGTCCCCTTCGGGTAAATAAATTTTAGTGGCAGTGGTACCGCCACTCCCTATTTTAATACTGCGAAGGAGGCAGTTGGCAGGCAACTCGTCGTTCGCAGTATATAAATATCCACTAATATTTATATTCCCGTAAGGGGACGTCCCGAAGGGGAAGGGGAAAGAAGCAGTCGCCTCCTTGCGAAAAGGTTTACTTGCCCGACCAGTGAAAAGCATGCTGTAAGATATAAATCTACCCTGAAAGGGATGCATTTCACCATAATACTATACAAATGGTGTTACCCTTTGAGGATCATAACGGTGCTACTGGAATATATGGTCTCTTCATGGATAGACGATAGCCATTTATTTACCCATTAAGGGGACATTAGTGGCCTGTCACTGCTCCTTACGAGACGCCAGTGGACGTTCGTCCTAGAAAATTTATGCGCTGCCTAGAAGCCCCAAAAGGGAAGTTTACTGACTGGTTAGAGCGTGCGCTAACAGGTTTAAATACTTCAATATGTATATTAGGACGCCGGTGGCAGTGGTACCGCCACTGCCACCGTCGGAGGACGTCCCTTACGGTATATTATATACTAGGATTTTAATACTCCGAAGGAGGCAGTGGCGGTACCACTGCCACTAATATTTATATTCCCGTAAGGGACGTCCTCCTTCGGAGTATGTAAACATTCTAAGTTTACTTGCCCAATATTTATATTAGGCAGTTGGCAGGCAACTGCTAGCTCTCCTCCTTCGGAGTATGTAAACATCGCAGTATATAAATATCCACTAATATTTATATTCCCGTAAGGGGACGTCCCGAAGGGGAAGGGGAAGGACGTCAGTGGCAGTTGCCTGCCAACTGCCTAGGCAAGTAAACTTAGGAGTATATAAATATAGGCAGTCGCGGTACCACTGCCACTGACGTCCTGCCAACTGCCTAGGCAAGTAAACTTAAGTGGCACTAAAATGCATTTGCCCGAAGGGGAAGGAGGACGCCAGTGGCAGTGGTACCGCCACTGCCTCCTTCGGAGTATTAAAATCCTAGTATGTAAATCTGCTAGCGCAGGAAATAAATTTTATTCTATTTATATACTCCGTTAGGAGGTAAGTAAACCCCTTCCCCTTCGGGACGTCAGTGCAGTTGCCTGCCAACTGCCTAATATAAATATTAGACCACTAAAGTTTGGCAACTGCCAACTGTTGTCCTTCGGAGGAAAAAAAATGGTTAACTCGCAAGCAGTTAACATAACTAAAGTTTGTTACTTTACCGAAGACGTTTACCCTTTCTCGGTTAAGGAGACGGAGACAGTTGCACTGTGACTGCCTAGTATAGCAATTTTGTTTTTGTTTATATGCTCGACAAAATGACTTTCATAAAAATATAAAGTAGTTAGCTAGTTATTTTATATCACTATAACTAGGGTTCTCAGAGGCACCGAAGTCACTTGTAAAAATAGTACTTTTTAACTTGTTTAATCTTCGTGTTCTTCAAAAGGATCACGTAATTTTTTTGAAGGTGGACCAAAACTAACATAAACTGAATAGCCAGTTACACTTAACAGAAGAAACCATAAAAAAAAGGTAAAGAAAAAAGCTGGACTTTCCATAGCTCATTTAATAATAAAATTATTCTCTTTTCAACATATCTCTTAGATAGTTCAAAAGACTTGACGACTGTGTCCCACATTTTTAAACAAAATTAATCTACTCAAAATTTTGCCCTGAGAAAGAATAACTTACTTCGTTTTTGCAGTAGCCATTCATGTCACTTTGAAACTGTCCTTACAAAGTTAAACATTAATTAAAAATTATTTAATTTTTATATAACAAATATTATATTAAATAAAAAATGAACAAAGAACTTCTAAGATCGTCTTTAGTGAGTAATTAAAGAGTTTTACTTACCAGACAAGGCAGTTTTTTCATTCTTTTAAAGCAGGCAGTTCTGAAGGGGAAAAGGGACTGCCTACTGCGGTCCTAGGTAAATACATTTTTATGCAATTTATTTCTTGTGCTAGTAGGTTTCTATACTCACAAGAAGCAACCCCTTGACGAGAGAACGTTATCCTCAGAGTATTTATAATCCTGAGAGGGAATGCACTGAAGAATATTTTCCTTATTTTTTACAGAAAGTAAATAAAATAGCGCTAATAACGCTTAATTCATTTAATCAATTATGGCAACAGGAACTTCTAAAGCTAAACCATCAAAAGTAAATTCAGACTTCCAAGAACCTGGTTTAGTTACACCATTAGGTACTTTATTACGTCCACTTAACTCAGAAGCAGGTAAAGTATTACCAGGCTGGGGTACAACTGTTTTAATGGCTGTATTTATCCTTTTATTTGCAGCATTCTTATTAATCATTTTAGAAATTTACAACAGTTCTTTAATTTTAGATGACGTTTCTATGAGTTGGGAAACTTTAGCTAAAGTTTCTTAATTTTATTTAACACAAACATAAAATATAAAACTGTTTGTTAAGGCTAGCTGCTAAGTCTTCTTTTCGCTAAGGTAAACTAAGCAACTCAACCATATTTATATTCGGCAGTGGCACCGCCAACTGCCACTGGCCTTCCGTTAAGATAAACGCGTggatctcacgtgACTAGTgtcgagtggtaccgccactgcctagtatataaatatcggcagttggcaggatatttatatactccgaaggaacttgttagccgataggcgaggcaactgccactaaaatttatttgcctcctaacggagcattaaaatccctaagtttacttgcccgtaaggggaaggggacgtccactaatatttatattaggcagttggcaggcaacaataaatacatttgtcccgtaaggggacgtcctgccaactgcctatggtagctattaagtatatatatatgaaaagtgtgtataaactaaactaaaataaaccaggtatggttaaccagatttattttagtttaaaaaaaaattagttgtttgagctagagttagttgaagctaagtctagaTTAACCGGTTCCTTTATCATCATCATCTTTGTAATCACTTCCACCGCCACCTGAGCCTTGAAAGTATAAGTTTTCACCGGTACCTAAACATTGGCTATAATATGGATTTAAAACTTGACATGTAGTTCCTGATGCACACACAGTTGGACCTGAATAACCAATACCACCACATTGACCGTAATGAGATTGTGTTGGTCCAGGAGAAGAACCTGTGGTAGTAGCAGGACGACGTGTTGTAGTGGTGCCAGGTGGATTTCCACCTGGTGGATTACCACCTGAAGGATCGCCTGTAGAGCCAATTGGACCAAATTTGATATTACTAAAAGTTACTTTTGCATTAGGACTTTGGCTTTCAACTTGAGCAGGTACACCACTTGAAGTTGAACATGAACCACGAACAGCACCAGGAGTGCTTGAAGTTTCGTTTGTAGGATATGTACTATCTAACCATAACATATTAGCATAATAGTCATCCCATAATGACATAACTAAAACCATACCACCTGATGTTGCTTTCTTGAATTGAGTTAAACCACCTTTATCTGAGAAGCTGCTACCACCAAATTCTGCTTCTTCTGCTGTACAATAATCGTCATTAAGGCCGTTACCAGAATAAGAACCTAATTCTGCATTTGGTTGTTGAAAAGTTACACCATTTTGCACGTAATAACGATTAATAGCACCGCTAGTTTCGAACTGTGTAACAACTGTTAACTTTTTAGTTGTATCTAATGTGAATGAACTTCCTGGTCCATAAAAAGATGTATTTCCTAAACGATATGGGTCCCAATCGCAGCCATCAGGATCACATGTACCACCATAACGGTTATCACTGTATGTTCCACCGCAACCGTCGCCTTCACAAATTTCTTGGCCAACGGTAGTACAAGGATGTGGAGTTAATGCTTCACTAATTGAATTAGCTTCCCAAATATCCATTTCAGAACAACAAGATCCGTGACCACCAATTCCAGTATTTGCATTATTACTACTTGGTTCCCAACCTTCCACGTTAGCTTGACCGTTAATAAACTTTAAATCACGAGGACACTGAGAATCACAATAGCCTGTTCCGTATTTAGCACCTGCTGTATTAGTAGGATATTTGCTTACACCGCCATCAGCGTCCATTGAAACGAAATAAAGAGCACCATTTAAACCACATGGTAATTGACTCACATCTACGTCGAAACTGAACTCATTACCTAATAATGTAAATTCTTGATAGGTTGTGTCACTTGCCATTAAGTATAAACGTGCGCCTACATTTTTTTGTGCTGATTGAGTCACGAAACCAATTGATAATGAGTTACCTGATGTAGTAACGCCGTAAGTTGAAGCGTAAGCTGCACCATCTAAACAACAATTTTTAGCACAAGTTTCGTTATCGGGACAAAGTGTTGATGACCAAGTATTACCGTCATAACAATTAGTTGAACTATTAGTGGCATGTGTCCAACGCCAGTTAGCATCAATTACTACAGAGCCAGTTTGTTGTGTACAAGTACCTCCTGAAGAACATTTTTGCCATGTTAATGGAGGATGAGTTTCAGATTGTAAGGTACATGCTGACTGTGCACGAGCAGTAGCTAAGAAAGCACTAATAACAGCAAGTTTACGATATGGTACCATatgcgtgtatctccaaaataaaaaaacaactcatcgttacgttaaatttattattatttaattttaatcattgtgtatttaatattataacttatataaaataaaattaaaaataagcattttttacacacatatttttaaataaatctttaaacgggttatatatagttatatatatgggactagaactgctttgtgcatagtcatcacaattattatattataaaccatgaataaaggttttattattatgatataaaaatgcataaaatttttataaattttgcaagtaaaatatataattaggaaaaaatttaaaatttaaaatgttagtcaagtttacaactaatacttttaattttgtattttaagtattggacatttttgtggaattaaatgtaccaaatatccatttaatttcatACTAGTgatatctacgtaatcgatgaattcgatcccatttttataactggatctcaaaatacctataaacccattgttcttctcttttagctctaagaacaatcaatttataaatatatttattattatgctataatataaatactatataaatacatttacctttttataaatacatttaccttttttttaatttgcatgattttaatgcttatgctatcttttttatttagtccataaaacctttaaaggaccttttcttatgggatatttatattttcctaacaaagcaatcggcgtcataaactttagttgcttacgacgcctgtggacgtcccccccttccccttacgggcaagtaaacttagggattttaatgcaataaataaatttgtcctcttcgggcaaatgaattttagtatttaaatatgacaagggtgaaccattacttttgttaacaagtgatcttaccactcactatttttgttgaattttaaacttatttaaaattctcgagaaagattttaaaaataaacttttttaatcttttatttattttttcttttttCGTATGGAATTGCCCAATATTATTCAACAATTTATCGGAAACAGCGTTTTAGAGCCAAATAAAATTGGTCAGTCGCCATCGGATGTTTATTCTTTTAATCGAAATAATGAAACTTTTTTTCTTAAGCGATCTAGCACTTTATATACAGAGACCACATACAGTGTCTCTCGTGAAGCGAAAATGTTGAGTTGGCTCTCTGAGAAATTAAAGGTGCCTGAACTCATCATGACTTTTCAGGATGAGCAGTTTGAATTTATGATCACTAAAGCGATCAATGCAAAACCAATTTCAGCGCTTTTTTTAACAGACCAAGAATTGCTTGCTATCTATAAGGAGGCACTCAATCTGTTAAATTCAATTGCTATTATTGATTGTCCATTTATTTCAAACATTGATCATCGGTTAAAAGAGTCAAAATTTTTTATTGATAACCAACTCCTTGACGATATAGATCAAGATGATTTTGACACTGAATTATGGGGAGACCATAAAACTTACCTAAGTCTATGGAATGAGTTAACCGAGACTCGTGTTGAAGAAAGATTGGTTTTTTCTCATGGCGATATCACGGATAGTAATATTTTTATAGATAAATTCAATGAAATTTATTTTTTAGACCTTGGTCGTGCTGGGTTAGCAGATGAATTTGTAGATATATCCTTTGTTGAACGTTGCCTAAGAGAGGATGCATCGGAGGAAACTGCGAAAATATTTTTAAAGCATTTAAAAAATGATAGACCTGACAAAAGGAATTATTTTTTAAAACTTGATGAATTGAATTGAttccaagcattatctaaaatactctgcaggcacgctagcttgtactcaagctcgtaacgaaggtcgtgaccttgctcgtgaaggtggcgacgtaattcgttcagcttgtaaatggtctccagaacttgctgctgcatgtgaagtttggaaagaaattaaattcgaatttgatactattgacaaactttaatttttatttttcatgatgtttatgtgaatagcataaacatcgtttttatttttatggtgtttaggttaaatacctaaacatcattttacatttttaaaattaagttctaaagttatcttttgtttaaatttgcctgtctttataaattacgatgtgccagaaaaataaaatcttagctttttattatagaatttatctttatgtattatatttttataagttataataaaagaaatagtaacatactaaagcggatgtagcgcgtttatcttaacggaaggaattcggcgcctacgtacccgggtcgcgaggatccACGCGTTAATAGCTCACTTTTCTTTAAATTTAATTTTTAATTTAAAGGTGTAAGCAAATTGCCTGACGAGAGATCCACTTAAAGGATGACAGTGGCGGGCTACTGCCTACTTCCCTCCGGGATAAAATTTATTTGAAAAACGTTAGTTACTTCCTAACGGAGCATTGACATCCCCATATTTATATTAGGACGTCCCCTTCGGGTAAATAAATTTTAGTGGACGTCCCCTTCGGGCAAATAAATTTTAGTGGACAATAAATAAATTTGTTGCCTGCCAACTGCCTAGGCAAGTAAACTTGGGAGTATTAAAATAGGACGTCAGTGGCAGTTGCCTGCCAACTGCCTATATTTATATACTGCGAAGCAGGCAGTGGCGGTACCACTGCCACTGGCGTCCTAATATAAATATTGGGCAACTAAAGTTTATAGCAGTATTAACATCCTATATTTATATACTCCGAAGGAACTTGTTAGCCGATAGGCGAGGCAACAAATTTATTTATTGTCCCGTAAAAGGATGCCTCCAGCATCGAAGGGGAAGGGGACGTCCTAGGCCATAAAACTAAAGGGAAATCCATAGTAACTGATGTTATAAATTTATAGACTCCAAAAAACAGCTGCGTTATAAATAACTTCTGTTAAATATGGCCAAGGGGACAGGGGCACTTTCAACTAAGTGTACATTAAAAATTGACAATTCAATTTTTTTTAATTATAATATATATTTAGTAAAATATAACAAAAAGCCCCCATCGTCTAGgtagaattccagctggcggccgccctatg 30GTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGCCCCGACACCC Endo-β-GCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACA glucanaseGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCAC insertionCGAAACGCGCGAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTC cassetteATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGG (pSE-3HB-AACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACA K-tD2:ATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAAC BD05)ATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCaagctcgcggccgcagtactCTGCAGATTTTATGCAAAATTAAAGTCTTGTGACAACAGCTTTCTCCTTAAGTGCAAATATCGCCCATTCTTTCCTCTTTTCGTATATAAATGCTGTAATAGTAGGATGTCGTACCCGTAAAGGTACGACATTGAATATTAATATACTCCTAAGTTTACTTTCCCAATATTTATATTAGGACGTCCCCTTCGGGTAAATAAATTTTAGTGGCAGTGGTACCGCCACTCCCTATTTTAATACTGCGAAGGAGGCAGTTGGCAGGCAACTCGTCGTTCGCAGTATATAAATATCCACTAATATTTATATTCCCGTAAGGGGACGTCCCGAAGGGGAAGGGGAAAGAAGCAGTCGCCTCCTTGCGAAAAGGTTTACTTGCCCGACCAGTGAAAAGCATGCTGTAAGATATAAATCTACCCTGAAAGGGATGCATTTCACCATAATACTATACAAATGGTGTTACCCTTTGAGGATCATAACGGTGCTACTGGAATATATGGTCTCTTCATGGATAGACGATAGCCATTTATTTACCCATTAAGGGGACATTAGTGGCCTGTCACTGCTCCTTACGAGACGCCAGTGGACGTTCGTCCTAGAAAATTTATGCGCTGCCTAGAAGCCCCAAAAGGGAAGTTTACTGACTCGTTAGAGCGTGCGCTAACAGGTTTAAATACTTCAATATGTATATTAGGACGCCGGTGGCAGTGGTACCGCCACTGCCACCGTCGGAGGACGTCCCTTACGGTATATTATATACTAGGATTTTAATACTCCGAAGGAGGCAGTGGCGGTACCACTGCCACTAATATTTATATTCCCGTAAGGGACGTCCTCCTTCGGAGTATGTAAACATTCTAAGTTTACTTGCCCAATATTTATATTAGGCAGTTGGCAGGCAACTGCTAGCTCTCCTCCTTCGGAGTATGTAAACATCGCAGTATATAAATATCCACTAATATTTATATTCCCGTAAGGGGACGTCCCGAAGGGGAAGGGGAAGGACGTCAGTGGCAGTTGCCTGCCAACTGCCTAGGCAAGTAAACTTAGGAGTATATAAATATAGGCAGTCGCGGTACCACTGCCACTGACGTCCTGCCAACTGCCTAGGCAAGTAAACTTAAGTGGCACTAAAATGCATTTGCCCGAAGGGGAAGGAGGACGCCAGTGGCAGTGGTACCGCCACTGCCTCCTTCGGAGTATTAAAATCCTAGTATGTAAATCTGCTAGCGCAGGAAATAAATTTTATTCTATTTATATACTCCGTTAGGAGGTAAGTAAACCCCTTCCCCTTCGGGACGTCAGTGCAGTTGCCTGCCAACTGCCTAATATAAATATTAGACCACTAAAGTTTGGCAACTGCCAACTGTTGTCCTTCGGAGGAAAAAAAATGGTTAACTCGCAAGCAGTTAACATAACTAAAGTTTGTTACTTTACCGAAGACGTTTACCCTTTCTCGGTTAAGGAGACGGAGACAGTTGCACTGTGACTGCCTAGTATAGCAATTTTGTTTTTGTTTATATGCTCGACAAAATGACTTTCATAAAAATATAAAGTAGTTAGCTAGTTATTTTATATCACTATAACTAGGGTTCTCAGAGGCACCGAAGTCACTTGTAAAAATAGTACTTTTTAACTTGTTTAATCTTCGTGTTCTTCAAAAGGATCACGTAATTTTTTTGAAGGTGGACCAAAACTAACATAAACTGAATAGCCAGTTACACTTAACAGAAGAAACCATAAAAAAAAGGTAAAGAAAAAAGCTGGACTTTCCATAGCTCATTTAATAATAAAATTATTCTCTTTTCAACATATCTCTTAGATAGTTCAAAAGACTTGACGACTGTGTCCCACATTTTTAAACAAAATTAATCTACTCAAAATTTTGCCCTGAGAAAGAATAACTTACTTCGTTTTGCAGTAGCCATTCATGTCACTTTGAAACTGTCCTTACAAAGTTAAACATTAATTAAAAATTATTTAATTTTTATATAACAAATATTATATTAAATAAAAAATGAACAAAGAACTTCTAAGATCGTCTTTAGTGAGTAATTAAAGAGTTTTACTTACCAGACAAGGCAGTTTTTTCATTCTTTTAAAGCAGGCAGTTCTGAAGGGGAAAAGGGACTGCCTACTGCGGTCCTAGGTAAATACATTTTTATGCAATTTATTTCTTGTGCTAGTAGGTTTCTATACTCACAAGAAGCAACCCCTTGACGAGAGAACGTTATCCTCAGAGTATTTATAATCCTGAGAGGGAATGCACTGAAGAATATTTTCCTTATTTTTTACAGAAAGTAAATAAAATAGCGCTAATAACGCTTAATTCATTTAATCAATTATGGCAACAGGAACTTCTAAAGCTAAACCATCAAAAGTAAATTCAGACTTCCAAGAACCTGGTTTAGTTACACCATTAGGTACTTTATTACGTCCACTTAACTCAGAAGCAGGTAAAGTATTACCAGGCTGGGGTACAACTGTTTTAATGGCTGTATTTATCCTTTTATTTGCAGCATTCTTATTAATCATTTTAGAAATTTACAACAGTTCTTTAATTTTAGATGACGTTTCTATGAGTTGGGAAACTTTAGCTAAAGTTTCTTAATTTTATTTAACACAAACATAAAATATAAAACTGTTTGTTAAGGCTAGCTGCTAAGTCTTCTTTTCGCTAAGGTAAACTAAGCAACTCAACCATATTTATATTCGGCAGTGGCACCGCCAACTGCCACTGGCCTTCCGTTAAGATAAACGCGTggatctcacgtgACTAGTgtcgagtggtaccgccactgcctagtatataaatatcggcagttggcaggatatttatatactccgaaggaacttgttagccgataggcgaggcaactgccactaaaatttatttgcctcctaacggagcattaaaatccctaagtttacttgcccgtaaggggaaggggacgtccactaatatttatattaggcagttggcaggcaacaataaatacatttgtcccgtaaggggacgtcctgccaactgcctatggtagctattaagtatatatatatgaaaagtgtgtataaactaaactaaaataaaccaggtatggttaaccagatttattttagtttaaaaaaaaattagttgtttgagctagagttagttgaagctaagtctagaTTAACCGGTTCCTTTATCATCATCATCTTTGTAATCACTTCCACCGCACCTGAGCCTTGAAAGTATAAGTTTTCACCGGTACCCTTGCGAGCTAAACAACTACTTACTAATGAAGTGTCTGTCCATGAGTTTCCAGAAGAAGTAGGGGTTTCGGTTAATACATAAGTTGAATCAAAAGAACCAGCACCCCAACCTACATAACCTAAGTACACGTCAGAGTTTTGATTTAAGTACTGAATTTGTTGACACATGTCTTGGATACAACTCTGCACGTTTCCACCACCTGTTTCAGTTAAAATGGCTTGGCGATTATTTTGACGTAACCAAGTTGCTAAAGGACTAAAAGCACCATCAATATTATTTGTGGTACATTCGGCGTGTGTTCCGCTATTATCAGAATCAAGATATTTATGTACATCGAATATTAAGTTAGTGGTACTACCGTCTGGGTTAGTCACTTGACTAAGAGCAGCTGCGCTACCGTCAGAAATAAAAGCGCCGGCAGATTGCCAATCGTTGCCTGGTAAACTAATGAATTGTGATGTTGCACCAGCATTACGAATAGCAGTTACTACTTCTTGCACAGTTGCAGCCCAAGTATTTATGTTCACATCGTGAGGTTCATTCATAATACCGAACCAAACACGTGATTGACTAGCATATTTTGAAGCTAATTGGCTCCATAATGATGTAAATTGAGCATTAGTTGGACCACCTTGACCAATAATACCACCGTTCCAACGGGCATAATTATGAATATCAACAATACAATAGGCACCTAAAGATAAGCAACCTTGTACTAATTGATCATATTTACTAATTGATGTACTATCTAAGTTACCACCTAAATTGTTGTTAACTAAGTATTGCCAGCCCACTGGTAAACGGAAAATAGTCATACCATCTTCATTTACAAAGTGTTGCATTTGACCAATGCCATCTGGATAATTGTTTGAGCCAGTAAAATTTTTTAAAGGGGGATAAACTTTACTGGTAACACATGTACCATCGGTAGTACAACCAAAATCGAAACCTGCAATATTAACACCAGCGAAACGTACTCCAGAAGATGTAGGAGGTGTGCTGCTAGAAGTGCTAGTAGCACGAGTTGTTGTAGTTGGACCTGAAGGAGGGCGAGTTGATGTTGTTATAGTTGTTGCACCTGGAATACATTGAGCATAGTAAGGATTTAAGGTACTACATGCTGAGCCAGGAGCACAATTGGTAGGACCAGACCAACCAATACCACCACACTGACCCCAAACAGTCTGTTGAGCAACAGCACCACCATATAAGATAGATGCAGCAAGTAATAATGGTGCTACGCTTTTGTTTGGTACCATatgcgtgtatctccaaaataaaaaaacaactcatcgttacgttaaatttattattatttaattttaatcattgtgtatttaatattataacttatataaaataaaattaaaaataagcattttttacacacatatttttaaataaatctttaaacgggttatatatagttatatatatgggactagaactgctttgtgcatagtcatcacaattattatattataaaccatgaataaaggttttattattatgatataaaaatgcataaaatttttataaattttgcaagtaaaatatataattaggaaaaaatttaaaatttaaaatgttagtcaagtttacaactaatacttttaattttgtattttaagtattggacattttgtggaattaaatgtaccaaatatccatttaatttcatACTAGTgatatctacgtaatcgatgaattcgatcccatttttataactggatctcaaaatacctataaacccattgttcttctcttttagctctaagaacaatcaatttataaatatatttattattatgctataatataaatactatataaatacatttacctttttataaatacatttaccttttttttaatttgcatgattttaatgcttatgctatcttttttatttagtccataaaaccttaaaggaccttttcttatgggatatttatattttcctaacaaagcaatcggcgtcataaactttagttgcttacgacgcctgtggacgtcccccccttccccttacgggcaagtaaacttagggattttaatgcaataaataaatttgtcctcttcgggcaaatgaattttagtatttaaatatgacaagggtgaaccattacttttgttaacaagtgatcttaccactcactatttttgttgaattttaaacttatttaaaattctcgagaaagattttaaaaataaacttttttaatcttttatttattttttcttttttCGTATGGAATTGCCCAATATTATTCAACAATTTATCGGAAACAGCGTTTTAGAGCCAAATAAAATTGGTCAGTCGCCATCGGATGTTTATTCTTTTAATCGAAATAATGAAACTTTTTTTCTTAAGCGATCTAGCACTTTATATACAGAGACCACATACAGTGTCTCTCGTGAAGCGAAAATGTTGAGTTGGCTCTCTGAGAAATTAAAGGTGCCTGAACTCATCATGACTTTTCAGGATGAGCAGTTTGAATTTATGATCACTAAAGCGATCAATGCAAAACCAATTTCAGCGCTTTTTTTAACAGACCAAGAATTGCTTGCTATCTATAAGGAGGCACTCAATCTGTTAAATTCAATTGCTATTATTGATTGTCCATTTATTTCAAACATTGATCATCGGTTAAAAGAGTCAAAATTTTTTATTGATAACCAACTCCTTGACGATATAGATCAAGATGATTTTGACACTGAATTATGGGGAGACCATAAAACTTACCTAAGTCTATGGAATGAGTTAACCGAGACTCGTGTTGAAGAAAGATTGGTTTTTTCTCATGGCGATATCACGGATAGTAATATTTTTATAGATAAATTCAATGAAATTTATTTTTTAGACCTTGGTCGTGCTGGGTTAGCAGATGAATTTGTAGATATATCCTTTGTTGAACGTTGCCTAAGAGAGGATGCATCGGAGGAAACTGCGAAAATATTTTTAAAGCATTTAAAAAATGATAGACCTGACAAAAGGAATTATTTTTTAAAACTTGATGAATTGAATTGAttccaagcattatctaaaatactctgcaggcacgctagcttgtactcaagctcgtaacgaaggtcgtgaccttgctcgtgaaggtggcgacgtaattcgttcagcttgtaaatggtctccagaacttgctgctgcatgtgaagtttggaaagaaattaaattcgaatttgatactattgacaaactttaatttttatttttcatgatgtttatgtgaatagcataaacatcgttttatttttatggtgtttaggttaaatacctaaacatcattttacatttttaaaattaagttctaaagttatcttttgtttaaatttgcctgctttataaattacgatgtgccagaaaaataaaatcttagctttttattatagaatttatctttatgtattatattttataagttataataaaagaaatagtaacatactaaagcggatgtagcgcgtttatcttaacggaaggaattcggcgcctacgtacccgggtcgcgaggatccACGCGTTAATAGCTCACTTTTCTTTAAATTTAATTTTTAATTTAAAGGTGTAAGCAAATTGCCTGACGAGAGATCCACTTAAAGGATGACAGTGGCGGGCTACTGCCTACTTCCCTCCGGGATAAAATTTATTTGAAAAACGTTAGTTACTTCCTAACGGAGCATTGACATCCCCATATTTATATTAGGACGTCCCCTTCGGGTAAATAAATTTTAGTGGACGTCCCCTTCGGGCAAATAAATTTTAGTGGACAATAAATAAATTTGTTGCCTGCCAACTGCCTAGGCAAGTAAACTTGGGAGTATTAAAATAGGACGTCAGTGGCAGTTGCCTGCCAACTGCCTATATTTATATACTGCGAAGCAGGCAGTGGCGGTACCACTGCCACTGGCGTCCTAATATAAATATTGGGCAACTAAAGTTTATAGCAGTATTAACATCCTATATTTATATACTCCGAAGGAACTTGTTAGCCGATAGGCGAGGCAACAAATTTATTTATTGTCCCGTAAAAGGATGCCTCCAGCATCGAAGGGGAAGGGGACGTCCTAGGCCATAAAACTAAAGGGAAATCCATAGTAACTGATGTTATAAATTTATAGACTCCAAAAAACAGCTGCGTTATAAATAACTTCTGTTAAATATGGCCAAGGGGACAGGGGCACTTTCAACTAAGTGTACATTAAAAATTGACAATTCAATTTTTTTTAATTATAATATATATTTAGTAAAATATAACAAAAAGCCCCCATCGTCTAGgtagaattccagctggcggccgccctatg 31GTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGCCCCGACACCC Endo-GCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACA xylanaseGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCAC insertionCGAAACGCGCGAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTC cassetteATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGG (pSE-3HB-AACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACA K-tD2:ATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAAC BD11)ATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCaagctcgcggccgcagtactCTGCAGATTTTATGCAAAATTAAAGTCTTGTGACAACAGCTTTCTCCTTAAGTGCAAATATCGCCCATTCTTTCCTCTTTTCGTATATAAATGCTGTAATAGTAGGATGTCGTACCCGTAAAGGTACGACATTGAATATTAATATACTCCTAAGTTTACTTTCCCAATATTTATATTAGGACGTCCCCTTCGGGTAAATAAATTTTAGTGGCAGTGGTACCGCCACTCCCTATTTTAATACTGCGAAGGAGGCAGTTGGCAGGCAACTCGTCGTTCGCAGTATATAAATATCCACTAATATTTATATTCCCGTAAGGGGACGTCCCGAAGGGGAAGGGGAAAGAAGCAGTCGCCTCCTTGCGAAAAGGTTTACTTGCCCGACCAGTGAAAAGCATGCTGTAAGATATAAATCTACCCTGAAAGGGATGCATTTCACCATAATACTATACAAATGGTGTTACCCTTTGAGGATCATAACGGTGCTACTGGAATATATGGTCTCTTCATGGATAGACGATAGCCATTTATTTACCCATTAAGGGGACATTAGTGGCCTGTCACTGCTCCTTACGAGACGCCAGTGGACGTTCGTCCTAGAAAATTTATGCGCTGCCTAGAAGCCCCAAAAGGGAAGTTTACTGACTCGTTAGAGCGTGCGCTAACAGGTTTAAATACTTCAATATGTATATTAGGACGCCGGTGGCAGTGGTACCGCCACTGCCACCGTCGGAGGACGTCCCTTACGGTATATTATATACTAGGATTTTAATACTCCGAAGGAGGCAGTGGCGGTACCACTGCCACTAATATTTATATTCCCGTAAGGGACGTCCTCCTTCGGAGTATGTAAACATTCTAAGTTTACTTGCCCAATATTTATATTAGGCAGTTGGCAGGCAACTGCTAGCTCTCCTCCTTCGGAGTATGTAAACATCGCAGTATATAAATATCCACTAATATTTATATTCCCGTAAGGGGACGTCCCGAAGGGGAAGGGGAAGGACGTCAGTGGCAGTTGCCTGCCAACTGCCTAGGCAAGTAAACTTAGGAGTATATAAATATAGGCAGTCGCGGTACCACTGCCACTGACGTCCTGCCAACTGCCTAGGCAAGTAAACTTAAGTGGCACTAAAATGCATTTGCCCGAAGGGGAAGGAGGACGCCAGTGGCAGTGGTACCGCCACTGCCTCCTTCGGAGTATTAAAATCCTAGTATGTAAATCTGCTAGCGCAGGAAATAAATTTTATTCTATTTATATACTCCGTTAGGAGGTAAGTAAACCCCTTCCCCTTCGGGACGTCAGTGCAGTTGCCTGCCAACTGCCTAATATAAATATTAGACCACTAAAGTTTGGCAACTGCCAACTGTTGTCCTTCGGAGGAAAAAAAATGGTTAACTCGCAAGCAGTTAACATAACTAAAGTTTGTTACTTTACCGAAGACGTTTACCCTTTCTCGGTTAAGGAGACGGAGACAGTTGCACTGTGACTGCCTAGTATAGCAATTTTGTTTTTGTTTATATGCTCGACAAAATGACTTTCATAAAAATATAAAGTAGTTAGCTAGTTATTTTATATCACTATAACTAGGGTTCTCAGAGGCACCGAAGTCACTTGTAAAAATAGTACTTTTTAACTTGTTTAATCTTCGTGTTCTTCAAAAGGATCACGTAATTTTTTTGAAGGTGGACCAAAACTAACATAAACTGAATAGCCAGTTACACTTAACAGAAGAAACCATAAAAAAAAGGTAAAGAAAAAAGCTGGACTTTCCATAGCTCATTTAATAATAAAATTATTCTCTTTTCAACATATCTCTTAGATAGTTCAAAAGACTTGACGACTGTGTCCCACATTTTTAAACAAAATTAATCTACTCAAAATTTTGCCCTGAGAAAGAATAACTTACTTCGTTTTTGCAGTAGCCATTCATGTCACTTTGAAACTGTCCTTACAAAGTTAAACATTAATTAAAAATTATTTAATTTTTATATAACAAATATTATATTAAATAAAAAATGAACAAAGAACTTCTAAGATCGTCTTTAGTGAGTAATTAAAGAGTTTTACTTACCAGACAAGGCAGTTTTTTCATTCTTTTAAAGCAGGCAGTTCTGAAGGGGAAAAGGGACTGCCTACTGCGGTCCTAGGTAAATACATTTTTATGCAATTTATTTCTTGTGCTAGTAGGTTTCTATACTCACAAGAAGCAACCCCTTGACGAGAGAACGTTATCCTCAGAGTATTTATAATCCTGAGAGGGAATGCACTGAAGAATATTTTCCTTATTTTTTACAGAAAGTAAATAAAATAGCGCTAATAACGCTTAATTCATTTAATCAATTATGGCAACAGGAACTTCTAAAGCTAAACCATCAAAAGTAAATTCAGACTTCCAAGAACCTGGTTTAGTTACACCATTAGGTACTTTATTACGTCCACTTAACTCAGAAGCAGGTAAAGTATTACCAGGCTGGGGTACAACTGTTTTAATGGCTGTATTTATCCTTTTATTTGCAGCATTCTTATTAATCATTTTAGAAATTTACAACAGTTCTTTAATTTTAGATGACGTTTCTATGAGTTGGGAAACTTTAGCTAAAGTTTCTTAATTTTATTTAACACAAACATAAAATATAAAACTGTTTGTTAAGGCTAGCTGCTAAGTCTTCTTTTCGCTAAGGTAAACTAAGCAACTCAACCATATTTATATTCGGCAGTGGCACCGCCAACTGCCACTGGCCTTCCGTTAAGATAAACGCGTggatctcacgtgACTAGTgtcgagtggtaccgccactgcctagtatataaatatcggcagttggcaggatatttatatactccgaaggaacttgttagccgataggcgaggcaactgccactaaaatttatttgcctcctaacggagcattaaaatccctaagtttacttgcccgtaaggggaaggggacgtccactaatatttatattaggcagttggcaggcaacaataaatacatttgtcccgtaaggggacgtcctgccaactgcctatggtagctattaagtatatatatatgaaaagtgtgtataaactaaactaaaataaaccaggtatggttaaccagatttattttagtttaaaaaaaaattagttgtttgagctagagttagttgaagctaagtctagaTTAACCGGTTCCTTTATCATCATCATCTTTGTAATCACTTCCACCGCCACCTGAGCCTTGAAAGTATAAGTTTTCACCGGTACCGCTAACAGTGATAGAAGCACTACCTGATGAAAAATAACCTTCAACAGCTACAATTTGATAGTCCATTGTACCTAATGTTAAACCTTGTTGAGCCCATGCATTAAAGTGGTTTGCTGTATTAACACTACCACTTGAACGATGATTACGTCTTACACTCCAGTATTGGTAGAAAGTGGCAGTTCCAATTATAGATGGTTGATTTACGCGTTGAGTACGATAAATATCATAAACTGATCCATCTGAAGTAACTTCACCTAATTTAGTAGCACCTGTTGAAGGGTTGTATGTACCAAAGTTCTCTACAATATAATATTCAATTAATGGGTTACGGCTCCAACCGTATACACTTAAATAAGAATTACCATTAGGGTTGTAACTACCAGAGAAATTGATTACCTTATTCTTTGTACCAGGTTGCCAACCTTTTCCTCCAACAAAATTGCCTGAGTTACTCCAATTTACACTAAATTGACCACCAGGTCCATTAGTATATGTAACACCACCGTGTCCATCATTCCAGTAAGAATAAAAGTAACCGTTATTGTAACCTGTACCTGGTTGAATTGTTTGACGTTTTTCTACTGCAACTGATTCCACTTCAGCAGCTGGACGGCAACTTGCACGTGAAGGTGGAGATGCTGCTAAAAGACTTGTGAAAGATACTGGTACCATatgcgtgtatctccaaaataaaaaaacaactcatcgttacgttaaatttattattatttaattttaatcattgtgtatttaatattataacttatataaaataaaattaaaaataagcattttttacacacatatttttaaataaatctttaaacgggttatatatagttatatatatgggactagaactgctttgtgcatagtcatcacaattattatattataaaccatgaataaaggttttattattatgatataaaaatgcataaaatttttataaattttgcaagtaaaatatataattaggaaaaaatttaaaatttaaaatgttagtcaagtttacaactaatacttttaattttgtattttaagtattggacatttttgtggaattaaatgtaccaaatatccatttaatttcatACTAGTgatatctacgtaatcgatgaattcgatcccatttttataactggatctcaaaatacctataaacccattgttcttctcttttagctctaagaacaatcaatttataaatatatttattattatgctataatataaatactatataaatacatttacctttttataaatacatttaccttttttttaatttgcatgattttaatgcttatgctatcttttttatttagtccataaaacctttaaaggaccttttcttatgggatatttatattttcctaacaaagcaatcggcgtcataaactttagttgcttacgacgcctgtggacgtcccccccttccccttacgggcaagtaaacttagggattttaatgcaataaataaatttgtcctcttcgggcaaatgaattttagtatttaaatatgacaagggtgaaccattacttttgttaacaagtgatcttaccactcactatttttgttgaattttaaacttatttaaaattctcgagaaagattttaaaaataaacttttttaatcttttatttattttttcttttttCGTATGGAATTGCCCAATATTATTCAACAATTTATCGGAAACAGCGTTTTAGAGCCAAATAAAATTGGTCAGTCGCCATCGGATGTTTATTCTTTTAATCGAAATAATGAAACTTTTTTTCTTAAGCGATCTAGCACTTTATATACAGAGACCACATACAGTGTCTCTCGTGAAGCGAAAATGTTGAGTTGGCTCTCTGAGAAATTAAAGGTGCCTGAACTCATCATGACTTTTCAGGATGAGCAGTTTTGAATTTATGATCACTAAAGCGATCAATGCAAAACCAATTTCAGCGCTTTTTTTAACAGACCAAGAATTGCTTGCTATCTATAAGGAGGCACTCAATCTGTTAAATTCAATTGCTATTATTGATTGTCCATTTATTTCAAACATTGATCATCGGTTAAAAGAGTCAAAATTTTTTATTGATAACCAACTCCTTGACGATATAGATCAAGATGATTTTGACACTGAATTATGGGGAGACCATAAAACTTACCTAAGTCTATGGAATGAGTTAACCGAGACTCGTGTTGAAGAAAGATTGGTTTTTTCTCATGGCGATATCACGGATAGTAATATTTTTATAGATAAATTCAATGAAATTTATTTTTTAGACCTTGGTCGTGCTGGGTTAGCAGATGAATTTGTAGATATATCCTTTGTTGAACGTTGCCTAAGAGAGGATGCATCGGAGGAAACTGCGAAAATATTTTTAAAGCATTTAAAAAATGATAGACCTGACAAAAGGAATTATTTTTTAAAACTTGATGAATTGAATTGAttccaagcattatctaaaatactctgcaggcacgctagcttgtactcaagctcgtaacgaaggtcgtgaccttgctcgtgaaggtggcgacgtaattcgttcagcttgtaaatggtctccagaacttgctgctgcatgtgaagtttggaaagaaattaaattcgaatttgatactattgacaaactttaatttttatttttcatgatgtttatgtgaatagcataaacatcgtttttatttttatggtgtttaggttaaatacctaaacatcattttacatttttaaaattaagttctaaagttatcttttgtttaaatttgcctgtctttataaattacgatgtgccagaaaaataaaatcttagctttttattatagaatttatctttatgtattatattttataagttataataaaagaaatagtaacatactaaagcggatgtagcgcgtttatcttaacggaaggaattcggcgcctacgtacccgggtcgcgaggatccACGCGTTAATAGCTCACTTTTCTTTAAATTTAATTTTTAATTTAAAGGTGTAAGCAAATTGCCTGACGAGAGATCCACTTAAAGGATGACAGTGGCGGGCTACTGCCTACTTCCCTCCGGGATAAAATTTATTTGAAAAACGTTAGTTACTTCCTAACGGAGCATTGACATCCCCATATTTATATTAGGACGTCCCCTTCGGGTAAATAAATTTTAGTGGACGTCCCCTTCGGGCAAATAAATTTTAGTGGACAATAAATAAATTTGTTGCCTGCCAACTGCCTAGGCAAGTAAACTTGGGAGTATTAAAATAGGACGTCAGTGGCAGTTGCCTGCCAACTGCCTATATTTATATACTGCGAAGCAGGCAGTGGCGGTACCACTGCCACTGGCGTCCTAATATAAATATTGGGCAACTAAAGTTTATAGCAGTATTAACATCCTATATTTATATACTCCGAAGGAACTTGTTAGCCGATAGGCGAGGCAACAAATTTATTTATTGTCCCGTAAAAGGATGCCTCCAGCATCGAAGGGGAAGGGGACGTCCTAGGCCATAAAACTAAAGGGAAATCCATAGTAACTGATGTTATAAATTTATAGACTCCAAAAAACAGCTGCGTTATAAATAACTTCTGTTAAATATGGCCAAGGGGACAGGGGCACTTTCAACTAAGTGTACATTAAAAATTGACAATTCAATTTTTTTTAATTATAATATATATTTAGTAAAATATAACAAAAAGCCCCCATCGTCTAGgtagaattccagctggcggccgcc ctatg

Example 8 Construction of a C. reinhardtii Strain Transformed with aConstruct that does not Disrupt Photosynthetic Capability

In this example a nucleic acid encoding endo-β-glucanase from T. reeseiwas introduced into C. reinhardtii. Transforming DNA (SEQ ID NO. 28,Table 4) is shown graphically in FIG. 2B. In this instance the segmentlabeled “Transgene” is the endo-β-glucanase encoding gene (SEQ ID NO.16, Table 3), the segment which drives expression of the transgene(labeled 5′ UTR) is the 5′ UTR and promoter sequence for the psbC genefrom C. reinhardtii, the segment labeled 3′ UTR contains the 3′ UTR forthe psbA gene from C. reinhardtii, and the segment labeled “SelectionMarker” is the kanamycin resistance encoding gene from bacteria, whichis regulated by the 5′ UTR and promoter sequence for the atpA gene fromC. reinhardtii and the 3′ UTR sequence for the rbcL gene from C.reinhardtii. The transgene cassette is targeted to the 3HB locus of C.reinhardtii via the segments labeled “5′Homology” and “3′ Homology,”which are identical to sequences of DNA flanking the 3HB locus on the 5′and 3′ sides, respectively. All DNA manipulations carried out in theconstruction of this transforming DNA were essentially as described bySambrook et al., Molecular Cloning: A Laboratory Manual (Cold SpringHarbor Laboratory Press 1989) and Cohen et al., Meth. Enzymol. 297,192-208, 1998.

For these experiments, all transformations were carried out on C.reinhardtii strain 137c (mt+). Cells were grown to late log phase(approximately 7 days) in the presence of 0.5 mM 5-fluorodeoxyuridine inTAP medium (Gorman and Levine, Proc. Natl. Acad. Sci., USA 54:1665-1669,1965, which is incorporated herein by reference) at 23° C. underconstant illumination of 450 Lux on a rotary shaker set at 100 rpm.Fifty ml of cells were harvested by centrifugation at 4,000×g at 23° C.for 5 min. The supernatant was decanted and cells resuspended in 4 mlTAP medium for subsequent chloroplast transformation by particlebombardment (Cohen et al., supra, 1998). All transformations werecarried out under kanamycin selection (100 μg/ml), in which resistancewas conferred by the gene encoded by the segment in FIG. 2B labeled“Selection Marker.” (Chlamydomonas Stock Center, Duke University).

PCR was used to identify transformed strains. For PCR analysis, 10⁶algae cells (from agar plate or liquid culture) were suspended in 10 mMEDTA and heated to 95° C. for 10 minutes, then cooled to near 23° C. APCR cocktail consisting of reaction buffer, MgCl2, dNTPs, PCR primerpair(s) (Table 2 and shown graphically in FIG. 3B), DNA polymerase, andwater was prepared. Algae lysate in EDTA was added to provide templatefor reaction. Magnesium concentration is varied to compensate for amountand concentration of algae lysate in EDTA added. Annealing temperaturegradients were employed to determine optimal annealing temperature forspecific primer pairs.

To identify strains that contain the endo-β-glucanase gene, a primerpair was used in which one primer anneals to a site within the psbC5′UTR (SEQ ID NO. 10) and the other primer anneals within theendo-β-glucanase coding segment (SEQ ID NO. 3). Desired clones are thosethat yield a PCR product of expected size. To determine the degree towhich the endogenous gene locus is displaced (heteroplasmic vs.homoplasmic), a PCR reaction consisting of two sets of primer pairs wereemployed (in the same reaction). The first pair of primers amplifies theendogenous locus targeted by the expression vector (SEQ ID NOs. 13 and14). The second pair of primers (SEQ ID NOs. 6 and 7) amplifies aconstant, or control region that is not targeted by the expressionvector, so should produce a product of expected size in all cases. Thisreaction confirms that the absence of a PCR product from the endogenouslocus did not result from cellular and/or other contaminants thatinhibited the PCR reaction. Concentrations of the primer pairs arevaried so that both reactions work in the same tube; however, the pairfor the endogenous locus is 5× the concentration of the constant pair.The number of cycles used was >30 to increase sensitivity. The mostdesired clones are those that yield a product for the constant regionbut not for the endogenous gene locus. Desired clones are also thosethat give weak-intensity endogenous locus products relative to thecontrol reaction.

Results from this PCR on 96 clones were determined and the results areshown in FIG. 10. FIG. 10A shows PCR results using thetransgene-specific primer pair. As can be seen, multiple transformedclones are positive for insertion of the endo-β-glucanase gene. FIG. 10Bshows the PCR results using the primer pairs to differentiatehomoplasmic from heteroplasmic clones. As can be seen, multipletransformed clones are either homoplasmic or heteroplasmic to a degreein favor of incorporation of the transgene (e.g. numbers 67, 92).Unnumbered clones demonstrate the presence of wild-type psbA and, thus,were not selected for further analysis.

To ensure that the presence of the endo-β-glucanase-encoding gene led toexpression of the endo-β-glucanase protein, a Western blot wasperformed. Approximately 1×10⁸ algae cells were collected from TAP agarmedium and suspended in 0.5 ml of lysis buffer (750 mM Tris, pH=8.0, 15%sucrose, 100 mM beta-mercaptoethanol). Cells were lysed by sonication(5×30 sec at 15% power). Lysate was mixed 1:1 with loading buffer (5%SDS, 5% beta-mercaptoethanol, 30% sucrose, bromophenol blue) andproteins were separated by SDS-PAGE, followed by transfer to PVDFmembrane. The membrane was blocked with TBST+5% dried, nonfat milk at23° C. for 30 min, incubated with anti-FLAG antibody (diluted 1:1,000 inTBST+5% dried, nonfat milk) at 4° C. for 10 hours, washed three timeswith TBST, incubated with horseradish-linked anti-mouse antibody(diluted 1:10,000 in TBST+5% dried, nonfat milk) at 23° C. for 1 hour,and washed three times with TBST. Proteins were visualized withchemiluminescent detection. Results from multiple clones (FIG. 10C) showthat expression of the endo-β-glucanase gene in C. reinhardtii cellsresulted in production of the protein.

Similar results were seen (FIG. 11) with a similar construct containingthe β-glucosidase gene from T. reesei (SEQ ID NO. 23, Table 4). Theconstruct containing the endoxylanase gene is depicted in FIG. 2B. Inthis instance the segment labeled “Transgene” is the β-glucosidaseencoding gene (SEQ ID NO. 17, Table 3), the segment which drivesexpression of the transgene (labeled 5′ UTR) is the 5′ UTR and promotersequence for the psbC gene from C. reinhardtii, the segment labeled 3′UTR contains the 3′ UTR for the psbA gene from C. reinhardtii, and thesegment labeled “Selection Marker” is the kanamycin resistance encodinggene from bacteria, which is regulated by the 5′ UTR and promotersequence for the atpA gene from C. reinhardtii and the 3′ UTR sequencefor the rbcL gene from C. reinhardtii. The transgene cassette istargeted to the 3HB locus of C. reinhardtii via the segments labeled“5′Homology” and “3′ Homology,” which are identical to sequences of DNAflanking the 3HB locus on the 5′ and 3′ sides, respectively. All DNAmanipulations carried out in the construction of this transforming DNAwere essentially as described by Sambrook et al., Molecular Cloning: ALaboratory Manual (Cold Spring Harbor Laboratory Press 1989) and Cohenet al., Meth. Enzymol. 297, 192-208, 1998.

FIG. 11A shows PCR using the gene-specific primer pair. As can be seen,multiple transformed clones are positive for insertion of theβ-glucosidase gene. FIG. 11B shows the PCR results using the primerpairs to differentiate homoplasmic from heteroplasmic clones. As can beseen, multiple transformed clones are either homoplasmic orheteroplasmic to a degree in favor of incorporation of the transgene(e.g. numbers 16, 64). Unnumbered clones demonstrate the presence ofwild-type psbA and, thus, were not selected for further analysis.Western blot analysis demonstrating protein expression is demonstratedin FIG. 11C.

Example 9 Construction of a Cyanobacteria Strain Expressing a BiomassDegrading Enzyme Construct that does not Disrupt PhotosyntheticCapability

In this example, a construct is made which is capable of insertion intoa selected cyanobacteria species (e.g., Synechocystis sp. strainPCC6803, Synechococcus sp. strain PCC7942, Thermosynechococcus elongatesBP-1, and Prochloroccus marina). Examples of such constructs arerepresented graphically in FIG. 13. In addition to the transgene andregulatory sequences (e.g., promoter and terminator), typically, suchconstructs will contain a suitable selectable marker (e.g., anantibiotic resistance gene). The transgene may be any gene of interest,but is preferably a biomass degrading enzyme (e.g., a cellulolytic,hemicellulolytic, ligninolytic enzyme). A cassette, or portion of thevector, may be integrated into the host cell genome via homologousrecombination when the exogenous DNA to be inserted is flanked byregions which share homology to portions of the cyanobacterial genome.Alternately, the construct may be a self-replicating vector which doesnot integrate into the host cell genome, but stably or transientlytransforms the host cell. In some instances, regulatory elements,transgenes, and/or selectable markers may need to be biased to thepreferred codon usage of the host organism. All DNA manipulations arecarried out essentially as described by Sambrook et al., MolecularCloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press 1989)and Cohen et al., Meth. Enzymol. 297, 192-208, 1998.

Transformation of Synechocystis with a construct of the presentinvention can be carried out by any method known in the art. (See, e.g.,Dzelzkalns and Bogorad, J. Bacteriol. 165: 964-71 (1986)). For thisexample Synechocystis sp. strain 6803 is grown to a density ofapproximately 2×10⁸ cells per ml and harvested by centrifugation. Thecell pellet is re-suspended in fresh BG-11 medium (ATCC Medium 616) at adensity of 1×10⁹ cells per ml and used immediately for transformation.One-hundred microliters of these cells are mixed with 5 ul of amini-prep solution containing the construct and the cells are incubatedwith light at 30° C. for 4 hours. This mixture is then plated onto nylonfilters resting on BG-11 agar supplemented with TES pH 8.0 and grown for12-18 hours. The filters are then transferred to BG-11 agar+TES+5 ug/mlampicillin and allowed to grow until colonies appear, typically within7-10 days.

Colonies are then picked into BG-11 liquid media containing 5 ug/mlampicillin and grown for 5 days. The transformed cells are incubatedunder low light intensity for 1-2 days and thereafter moved to normalgrowth conditions. These cells are then transferred to BG-11 mediacontaining 10 ug/ml ampicilin and allowed to grow for 5 days. Cells werethen harvested for PCR analysis to determine the presence of theexogenous insert. Western blots may be performed (essentially asdescribed above) to determine expression levels of the protein(s)encoded by the inserted construct.

Example 10 Expression of Biomass Degrading Enzymes in Escherichia coli

In this example a nucleic acid encoding endo-β-glucanase from T. reeseiwas cloned into pET-21a using the NdeI and XhoI restriction sitespresent in both the gene and pET-21a. The resulting vector (SEQ ID NO.25, Table 4) was transformed into E. coli BL-21 cells. Expression wasinduced when cell density reached OD=0.6. Cells were grown at 30° C. for5 hours and then harvested. Purification was essentially as describedpreviously. Activity of the enzymes expressed in bacteria was determinedusing assays essentially as described in previous examples. The resultsof these analyses are shown in FIG. 17 (Lane 2).

Nucleic acids encoding exo-β-glucanase, β-glucosidase and endoxylanasewere also cloned into pET-21. The resulting vectors (SEQ ID NOs. 24, 26and 27, respectively, Table 4) were transformed into E. coli BL-21cells. Expression was induced when cell density reached OD=0.6. Cellswere grown at 30° C. for 5 hours and then harvested. Purification wasessentially as described previously. Activity of the enzymes expressedin bacteria was determined using assays essentially as described inprevious examples. The results of these analyses are shown in FIG. 17(Lane 1: exo-β-glucanase; Lane 3: β-glucosidase; and Lane 4:endoxylanase). Enzyme activity was also measured, essentially aspreviously described. Results, which are presented inbackground-subtracted values, are provided in Table 5.

TABLE 5 Enzyme activity of bacterially-produced biomass degradingenzymes Filter paper Enzyme Added assay β-glucosidase assay Xylanaseassay Control (TBS) 0.000 0.000 0.000 endo-β-glucanase 0.194 0.000 0.020β-glucosidase 0.006 0.525 0.000 endoxylanase 0.000 0.011 3.131

This data, along with the data shown in previous examples, demonstratesthat the enzymes encoded by the vectors described herein can befunctionally expressed by both algae and bacteria, despite the codonbias built into the sequences.

Various modifications, processes, as well as numerous structures thatmay be applicable herein will be apparent. Various aspects, features orembodiments may have been explained or described in relation tounderstandings, beliefs, theories, underlying assumptions, and/orworking or prophetic examples, although it will be understood that anyparticular understanding, belief, theory, underlying assumption, and/orworking or prophetic example is not limiting. Although the variousaspects and features may have been described with respect to variousembodiments and specific examples herein, it will be understood that anyof same is not limiting with respect to the full scope of the appendedclaims or other claims that may be associated with this application.

1. A method of screening a transformed non-vascular photosyntheticorganism, comprising: amplifying a first endogenous chloroplast nucleicacid sequence of said organism using a first primer pair, wherein saidfirst endogenous chloroplast nucleic acid sequence is a target of anexpression vector for transforming said organism and one primer of saidfirst primer pair anneals in said first endogenous chloroplast nucleicacid sequence that is the target of said expression vector; amplifying asecond endogenous chloroplast nucleic acid sequence of said organismusing a second primer pair, wherein said second endogenous chloroplastnucleic acid sequence is a control sequence that is not said target ofan expression vector for transforming said organism; and determining theplasmic state of said organism based on results from amplification ofsaid first sequence and second sequence, wherein said first and secondprimer pairs are different.
 2. The method of claim 1, wherein saidamplifying of said first and second nucleic acid sequences is performedsimultaneously.
 3. The method of claim 1, wherein said first primer pairamplifies a region containing an untranslated region (UTR) and a codingregion of said first endogenous chloroplast nucleic acid sequence. 4.The method of claim 3, wherein said untranslated region is a 5′untranslated region.
 5. The method of claim 1, further comprising thestep of amplifying a third chloroplast nucleic acid sequence.
 6. Themethod of claim 5, wherein said third nucleic acid is an exogenousnucleic acid sequence.
 7. The method of claim 5, wherein said amplifyingsaid third nucleic acid is performed concurrently in a second separatereaction from said amplifying of said first or second nucleic acidsequence.
 8. The method of claim 5, wherein all amplifications areperformed in a single reaction.
 9. The method of claim 1, wherein saidplasmic state is homoplasmy.
 10. The method of claim 1, wherein saidnon-vascular photosynthetic organism is a microalga.
 11. The method ofclaim 1, wherein said organism comprises an exogenous nucleic acidsequence comprising at least one gene of interest and a selectablemarker.
 12. The method of claim 1, wherein said amplification of saidfirst nucleic acid sequence or said second nucleic acid sequence or bothcomprises more than 30 cycles of polymerase chain reaction (PCR).
 13. Amethod of producing a genetically-modified homoplasmic non-vascularphotosynthetic organism, comprising: transforming at least onechloroplast of said organism with an exogenous nucleic acid sequence;amplifying a first endogenous chloroplast nucleic acid sequence of saidorganism using a first primer pair, wherein said first endogenouschloroplast nucleic acid sequence is a target of an expression vectorfor genetically modifying said organism with said at least one exogenousnucleic acid sequence and one primer of said first primer pair annealsin said first endogenous chloroplast nucleic acid sequence that is saidtarget of said expression vector; amplifying a second endogenouschloroplast nucleic acid sequence of said organism using a second primerpair, wherein said second endogenous chloroplast nucleic acid sequenceis a control sequence that is not said target of an expression vectorfor genetically modifying said organism; determining the plasmic stateof said organism based on results from said amplifying of said firstendogenous chloroplast nucleic acid sequence and said second endogenouschloroplast nucleic acid sequence; and selecting a homoplasmic organism.14. The method of claim 13, wherein said first and second nucleic acidsequences are amplified in a single reaction.
 15. The method of claim13, wherein said first primer pair amplifies a region containing anuntranslated region (UTR) and a coding region of said first endogenouschloroplast nucleic acid sequence.
 16. The method of claim 15, whereinsaid untranslated region is a 5′ untranslated region.
 17. The method ofclaim 13, further comprising the step of amplifying a third chloroplastnucleic acid sequence.
 18. The method of claim 17, wherein said thirdnucleic acid sequence is at least partially from an exogenous nucleicacid.
 19. The method of claim 13, wherein said amplification of saidfirst nucleic acid sequence or said second nucleic acid sequence or bothcomprises more than 30 cycles of PCR.
 20. The method of claim 13,wherein said non-vascular photosynthetic organism is a microalga. 21.The method of claim 13, wherein said exogenous nucleic acid comprises atleast one gene of interest and a selectable marker.
 22. The method ofclaim 21, wherein said gene of interest encodes a biomass degradingenzyme.